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A Transcriptively Active Complex of APP with Fe65 and Histone Acetyltransferase Tip60
Abstract
Amyloid-beta precursor protein (APP), a widely expressed cell-surface protein, is cleaved in the transmembrane region by gamma-secretase. gamma-Cleavage of APP produces the extracellular amyloid beta-peptide of Alzheimer's disease and releases an intracellular tail fragment of unknown physiological function. We now demonstrate that the cytoplasmic tail of APP forms a multimeric complex with the nuclear adaptor protein Fe65 and the histone acetyltransferase Tip60. This complex potently stimulates transcription via heterologous Gal4- or LexA-DNA binding domains, suggesting that release of the cytoplasmic tail of APP by gamma-cleavage may function in gene expression.
... In a pioneer experiment designed to investigate the putative involvement of AICD in transcription Cao and Sudhof (2001) used a fusion protein consisting of the DNA-binding domain of the yeast Gal4 (or bacterial LexA) transcription factor and the C-terminal APP domain and showed that such protein could activate transcription from the Gal4-or LexA-dependent reporter plasmid, albeit to a small extent. Interestingly, cotransfection with the Fe65 protein highly elevated reporter gene activity when assayed in several cell types. ...
... Interestingly, cotransfection with the Fe65 protein highly elevated reporter gene activity when assayed in several cell types. Moreover, mutation within the Fe65 binding site abolished transcriptional activation mediated by the C-terminal domain of APP (Cao and Sudhof 2001). TIP60, a histone acetylotransferase identified as a binding partner of Fe65, did not show transactivation ability when assayed as a Gal4-fusion protein but appeared to act as a co-activator for the AICD-Fe65 complex. ...
... A simultaneous co-expression of APP (or AICD), Fe65 and Gal4-Tip60 led to dramatically enhanced expression of the reporter gene (over 100 times) while the presence of CP2, a transcription factor interacting with Fe65, had no effect. These results indicate that the AICD-Fe65-Tip60 triplet may play a role in activating Fig. 4. Schematic representation of the transcriptional protein complex containing AICD gene transcription (Cao and Sudhof 2001). An alternative model was proposed in which the C-terminal part of APP is required only to activate Fe65 in a membrane-dependent process making AICD nuclear translocation dispensable for the observed transcription activation (Cao and Sudhof 2004). ...
Amyloid Precursor Protein (APP) Intracellular Domain (AICD) is the product of APP processing realized by α- or β-secretases and γ-secretase. It was shown that AICD is able to interact with several proteins which regulate its stability and cellular localization. The Fe65 adaptor protein translocates AICD into nucleus where the APP-Fe65-Tip60 ternary complex may activate transcription of target genes. In the light of recent studies AICD seems to be another product of APP proteolysis endowed with important biological functions that may contribute to Alzheimer’s disease pathology.
... 37,38 AICD could translocate into the nucleus to regulate gene expression by forming a complex with Fe65 and Tip60. 39 In this study, we found that a motif within the transmembrane domain of CNTNAP2 was highly homologous to APP's γ-secretase cleavage site, suggesting that CNTNAP2 may undergo proteolytic cleavage. Our biochemical studies demonstrated that CNTNAP2 was first cleaved in extracellular juxtamembrane domain releasing a soluble extracellular fragment and a membrane-tethered 20KDa C-terminal fragment (CTF). ...
... CNTNAP2 undergoes proteolytic cleavage Proteolytic processing of many single transmembrane proteins such as Notch and APP 34,36,[39][40][41] to release the intracellular domain is required for their functions. We found that CNTNAP2 contains a VVIF motif within its transmembrane domain that is highly homologous to the VVIA of APP's γ-secretase cleavage site (Fig. 1a). ...
... CICD transcriptionally activates Necdin expression Some single transmembrane proteins, such as APP and Notch receptors, are proteolytically cleaved to produce intracellular domains, which translocate into the nucleus to transcriptionally regulate gene expression. 34,36,39 Previously by performing RNAsequencing analysis, 99 significantly down-regulated genes and 90 significantly up-regulated genes were identified in the hippocampus of Cntnap2 −/− mice. 32 Among these genes, Necdin in the Prader-Willi syndrome (PWS) chromosomal region is the most notably changed gene (Fig. 3a), and patients with PWS also present with autism-related symptoms. ...
As the most prevalent neurodevelopmental disorders in children, autism spectrum disorders (ASD) are characterized by deficits in language development, social interaction, and repetitive behaviors or inflexible interests. Contactin associated protein like 2 (CNTNAP2) , encoding a single transmembrane protein (CNTNAP2) with 1331 amino acid residues, is a widely validated ASD-susceptible gene. Cntnap2 -deficient mice also show core autism-relevant behaviors, including the social deficits and repetitive behavior. However, the cellular mechanisms underlying dysfunction CNTNAP2 and ASD remain elusive. In this study, we found a motif within the transmembrane domain of CNTNAP2 was highly homologous to the γ-secretase cleavage site of amyloid-β precursor protein (APP), suggesting that CNTNAP2 may undergo proteolytic cleavage. Further biochemical analysis indicated that CNTNAP2 is cleaved by γ-secretase to produce the CNTNAP2 intracellular domain (CICD). Virally delivery of CICD to the medial prefrontal cortex (mPFC) in Cntnap2 -deficient ( Cntnap2 −/− ) mice normalized the deficit in the ASD-related behaviors, including social deficit and repetitive behaviors. Furthermore, CICD promoted the nuclear translocation of calcium/calmodulin-dependent serine protein kinase (CASK) to regulate the transcription of genes, such as Prader Willi syndrome gene Necdin . Whereas Necdin deficiency led to reduced social interaction in mice, virally expression of Necdin in the mPFC normalized the deficit in social preference of Cntnap2 −/− mice. Our results thus reveal a critical function of CICD and highlight a role of the CNTNAP2-CASK-Necdin signaling pathway in ASD.
... This is a cellular process that is frequently implicated in important signaling pathways [50,51], also involves the nucleus. Among APP metabolites, the intracellular domain AICD, which is released into the cytosol, has been extensively studied regarding its function in nuclear signaling [52,53]. However, in the last decade, the nuclear function of Aβ42 has also been investigated, also showing DNA binding properties of this peptide. ...
... Cytosolic AICD is subjected to rapid degradation [54,55], which leads to a short half-life [56]. Nevertheless, binding with the adaptor protein Fe65 stabilizes it and promotes its nuclear translocation [52,56,57]. Fe65, also known as the Amyloid Beta Precursor Protein Binding Family B Member 1 (APBB1), is a multifunctional protein belonging to the Fe65 family with the related proteins Fe65L1 and Fe65L2. ...
... Fe65, also known as the Amyloid Beta Precursor Protein Binding Family B Member 1 (APBB1), is a multifunctional protein belonging to the Fe65 family with the related proteins Fe65L1 and Fe65L2. It was recognized as an APP [58] and AICD [52,59] binding partner in yeast two-hybrid screenings. Fe65 cytosolic/nuclear localization is modulated by APP binding [60] and phosphorylation [61], as well as by its own phosphorylation [62]. ...
Alzheimer’s disease (AD) is a neurodegenerative disorder representing the most common form of dementia. It is biologically characterized by the deposition of extracellular amyloid-β (Aβ) senile plaques and intracellular neurofibrillary tangles, constituted by hyperphosphorylated tau protein. The key protein in AD pathogenesis is the amyloid precursor protein (APP), which is cleaved by secretases to produce several metabolites, including Aβ and APP intracellular domain (AICD). The greatest genetic risk factor associated with AD is represented by the Apolipoprotein E ε4 (APOE ε4) allele. Importantly, all of the above-mentioned molecules that are strictly related to AD pathogenesis have also been described as playing roles in the cell nucleus. Accordingly, evidence suggests that nuclear functions are compromised in AD. Furthermore, modulation of transcription maintains cellular homeostasis, and alterations in transcriptomic profiles have been found in neurodegenerative diseases. This report reviews recent advancements in the AD players-mediated gene expression. Aβ, tau, AICD, and APOE ε4 localize in the nucleus and regulate the transcription of several genes, part of which is involved in AD pathogenesis, thus suggesting that targeting nuclear functions might provide new therapeutic tools for the disease.
... 4 AICD binds to the phosphotyrosine binding domain (PTB2) on Fe65 and is translocated into the nucleus and docked to Tip60, a chromatin-associated histone acetyltransferase, and the ternary complex promotes gene expression. [5][6][7][8] Numerous AICD target genes have been reported including glycogen synthase kinase-3b (GSK3B) 9 and stathmin 1, 7 mediating various cellular events from apoptosis to cytoskeletal dynamics. 6 These proteins form AICD-Fe65-Tip60 (AFT) complexes that are concentrated in spherical nuclear spots. ...
... Results d-Secretase-truncated APP but not full-length APP selectively associates with CEBPB AICD translocates into the nucleus and promotes gene expression. 5 To explore whether APP C586-695 (C110) from d-secretase cleavage possesses similar functions, we tested whether it directly interacts with the key transcription factor CEBPB, which mediates d-secretase expression. 19 We co-transfected GST-tagged APP, tau or a-syn and their d-secretase-truncated fragments with GFP-CEBPB into HEK293 cells, respectively. ...
... 4 Fe65 forms a transcriptionally active complex with APP/AICD in heterologous gene reporter systems and activates gene transcription. 5 The analogy of APP processing by c-secretase to Notch receptor signalling suggests a possible function for the AICD in the nuclear signalling. [43][44][45] The transactivation activity for AICD is dependent on APP-adaptor protein Fe65 46,47 and Fe65-binding nuclear histone acetyl transferase Tip60. ...
Amyloid-β precursor protein (APP) is sequentially cleaved by secretases and generates amyloid-β, the major components in senile plaques in Alzheimer’s disease. APP is upregulated in human Alzheimer’s disease brains. However, the molecular mechanism of how APP contributes to Alzheimer’s disease pathogenesis remains incompletely understood. Here we show that truncated APP C586-695 fragment generated by δ-secretase directly binds to CCAAT/enhancer-binding protein beta (CEBPB), an inflammatory transcription factor, and enhances its transcriptional activity, escalating Alzheimer’s disease-related gene expression and pathogenesis. The APP C586-695 fragment, but not full-length APP, strongly associates with CEBPB and elicits its nuclear translocation and augments the transcriptional activities on APP itself, MAPT (microtubule-associated protein tau), δ-secretase and inflammatory cytokine mRNA expression, finally triggering Alzheimer’s disease pathology and cognitive disorder in a viral overexpression mouse model. Blockade of δ-secretase cleavage of APP by mutating the cleavage sites reduces its stimulatory effect on CEBPB, alleviating amyloid pathology and cognitive dysfunctions. Clearance of APP C586-695 from 5xFAD mice by antibody administration mitigates Alzheimer’s disease pathologies and restores cognitive functions. Thus, in addition to the sequestration of amyloid-β, APP implicates in Alzheimer’s disease pathology by activating CEBPB upon δ-secretase cleavage.
... AICD produced through the non-amyloidogenic pathway undergo a rapid cytoplasmatic degradation through the endolysosomal system [45] and by the Insulin-Degrading Enzyme (IDE, a large zinc-binding protease of the M16 metalloprotease family) [46,47]. In contrast, AICD generated via the amyloidogenic pathway bind the Fe65 adaptor protein and translocate into the nucleus, where they associate with Tip60 to form the ATF complex [48]. The ATF complex acts as a transcription factor and regulates the expression of different APP-related genes, including APP itself (its own precursor), BACE1, GSK3B (coding for GSK-3β) and MME (coding for the Aβ-degrading enzyme Neprilysin, also known as Membrane Metallo-Endopeptidase (MME), Neutral Endopeptidase (NEP), cluster of differentiation 10 (CD10), and Common Acute Lymphoblastic Leukaemia Antigen (CALLA)) among the others [49][50][51]. ...
... Similarly to AICD, ALID1 and ALID2 have been proposed to play a functional role as transcriptional regulators [53], although the validity of these speculations is still controversial due to the lack of adequate animal models. associate with Tip60 to form the ATF complex [48]. The ATF complex acts as a transcription factor and regulates the expression of different APP-related genes, including APP itself (its own precursor), BACE1, GSK3B (coding for GSK-3β) and MME (coding for the Aβ-degrading enzyme Neprilysin, also known as Membrane Metallo-Endopeptidase (MME), Neutral Endopeptidase (NEP), cluster of differentiation 10 (CD10), and Common Acute Lymphoblastic Leukaemia Antigen (CALLA)) among the others [49][50][51]. ...
Amyloid Precursor Protein (APP) and its cleavage processes have been widely investigated in the past, in particular in the context of Alzheimer’s Disease (AD). Evidence of an increased expression of APP and its amyloidogenic-related cleavage enzymes, β-secretase 1 (BACE1) and γ-secretase, at the hit axon terminals following Traumatic Brain Injury (TBI), firstly suggested a correlation between TBI and AD. Indeed, mild and severe TBI have been recognised as influential risk factors for different neurodegenerative diseases, including AD. In the present work, we describe the state of the art of APP proteolytic processing, underlining the different roles of its cleavage fragments in both physiological and pathological contexts. Considering the neuroprotective role of the soluble APP alpha (sAPPα) fragment, we hypothesised that sAPPα could modulate the expression of genes of interest for AD and TBI. Hence, we present preliminary experiments addressing sAPPα-mediated regulation of BACE1, Isthmin 2 (ISM2), Tetraspanin-3 (TSPAN3) and the Vascular Endothelial Growth Factor (VEGFA), each discussed from a biological and pharmacological point of view in AD and TBI. We finally propose a neuroprotective interaction network, in which the Receptor for Activated C Kinase 1 (RACK1) and the signalling cascade of PKCβII/nELAV/VEGF play hub roles, suggesting that vasculogenic-targeting therapies could be a feasible approach for vascular-related brain injuries typical of AD and TBI.
... 51 The concept of protein folding disorder has its origins in the mid-19th century, when, in 1854, Rudolf Virchow coined 52 the term amyloid, from the Latin word "amylum" (starch), to describe a substance in the starchy brain bodies that 53 exhibited a chemical reaction resembling that of cellulose [5]. Indeed, a great number of protein folding diseases are 54 associated with the formation of highly organized fibrillar aggregates often described as amyloids [4]. This group of 55 protein folding diseases is also known as amyloidoses. ...
... [51] Intracellular C-terminal Domain (AICD) could possess signaling functions (see [52] and [53] for a review). Reporter 128 gene assays based on Gal4-fusion constructs suggested that AICD could act as transcriptional regulator [54,55], but the 129 controversy about the transcriptional activity of AICD and its target genes has only grown since then [56,57]. 2.1. ...
Most neurodegenerative diseases have the characteristics of proteinopathies, i.e. they cause lesions to appear in vulnerable regions of the nervous system, corresponding to protein aggregates that progressively spread through the neuronal network as the symptoms progress. Alzheimer's disease is one of these proteinopathies. It is characterized by two lesions, neurofibrillary tangles (NFTs) and senile plaques, formed essentially of amyloid peptides (Aβ). A combination of factors ranging from genetic mutations to age-related changes in the cellular context converge in this disease to accelerate Aβ deposition. Over the last two decades, numerous studies have attempted to elucidate how structural determinants of its precursor (APP) modify Aβ production, and to understand the processes leading to the formation of different Aβ aggregates; e.g. fibrils and oligomers. The synthesis proposed in this review indicates that the same motifs can control APP function and Aβ production essentially by regulating membrane dimerization, and subsequently Aβ aggregation processes. The distinct properties of these motifs and the cellular context regulate the APP conformation to trigger the transition to the amyloid pathology. This concept can be transposed to the study of other proteinopathies, providing a framework for improving our understanding of these mechanisms that devastate neuronal functions.
... APP is a membrane-bound protein, and AICD is released into the cytosol after -secretase cleavage at the -cleavage site. Of high relevance to our study, AICD has been implicated as a transcriptional regulator (11)(12)(13)(14). This activity is dependent on its ability to translocate to the nucleus, a process that requires its protection from degradation in the cytosol by docking proteins, notably Fe65. ...
... We previously identified APP phosphorylation at Thr 668 within the AICD as an LRRK2 substrate that contributes to LRRK2-mediated neurodegeneration (10). Given that AICD has been implicated as a transcriptional regulator (11)(12)(13), we hypothesized that AICD may regulate LRRK2 expression at the transcriptional level. To this end, we established an in vitro cell-based assay to evaluate the effect of AICD on LRRK2 transcription. ...
Gain-of-function mutations in the leucine-rich repeat kinase 2 ( LRRK2 ) gene are common in familial forms of Parkinson’s disease (PD), which is characterized by progressive neurodegeneration that impairs motor and cognitive function. We previously demonstrated that LRRK2-mediated phosphorylation of β-amyloid precursor protein (APP) triggers the production and nuclear translocation of the APP intracellular domain (AICD). Here, we connected LRRK2 to AICD in a feed-forward cycle that enhanced LRRK2-mediated neurotoxicity. In cooperation with the transcription factor FOXO3a, AICD promoted LRRK2 expression, thus increasing the abundance of LRRK2 that promotes AICD activation. APP deficiency in LRRK2 G2019S mice suppressed LRRK2 expression, LRRK2-mediated mitochondrial dysfunction, α-synuclein accumulation, and tyrosine hydroxylase (TH) loss in the brain, phenotypes associated with toxicity and loss of dopaminergic neurons in PD. Conversely, AICD overexpression increased LRRK2 expression and LRRK2-mediated neurotoxicity in LRRK2 G2019S mice. In LRRK2 G2019S mice or cultured dopaminergic neurons from LRRK2 G2019S patients, treatment with itanapraced reduced LRRK2 expression and was neuroprotective. Itanapraced showed similar effects in a neurotoxin-induced PD mouse model, suggesting that inhibiting the AICD may also have therapeutic benefits in idiopathic PD. Our findings reveal a therapeutically targetable, feed-forward mechanism through which AICD promotes LRRK2-mediated neurotoxicity in PD.
... The generation of amyloid beta through APP cleavage leads to altered downstream signalling, activity and production of HDAC4, SOX2 and F2 through changes in caspase-3 (CASP3) [415,416], JUN [417,418] and thrombospondin-1 (THBS1) [419,420], respectively ( Figure 20B). Cleavage of APP generates a cytosolic fragment, AICD, which forms a transcriptionally active complex with TIP60 and the transcription factor FE65 [421]. AICD also modulates the ubiquitin-proteasome system (UPS) via UBE2N [422], to change downstream signalling induced by TNF [423] ( Figure 20B). ...
... Arc's activation of the γ-secretase PSEN1 to promote cleavage of APP not only increases amyloid beta load, but also results in an increased level of the APP intracellular domain (AICD) [214,447]. AICD forms a complex with TIP60/Kat5 to alter transcriptional activity crucial for AD progression [421,[448][449][450][451][452][453] (Figure 20B). This AICD-TIP60 interaction is disrupted by NICD, formed when Arc activates NOTCH1 [381], thereby downregulating AICD signalling while promoting NICD signalling [454,455] (Figure 20B). ...
The immediate early gene Arc is a master regulator of synaptic function and a critical determinant of memory consolidation. Here, we show that Arc interacts with dynamic chromatin and closely associates with histone markers for active enhancers and transcription in cultured rat hippocampal neurons. Both these histone modifications, H3K27Ac and H3K9Ac, have recently been shown to be upregulated in late-onset Alzheimer’s disease (AD). When Arc induction by pharmacological network activation was prevented using a short hairpin RNA, the expression profile was altered for over 1900 genes, which included genes associated with synaptic function, neuronal plasticity, intrinsic excitability, and signalling pathways. Interestingly, about 100 Arc-dependent genes are associated with the pathophysiology of AD. When endogenous Arc expression was induced in HEK293T cells, the transcription of many neuronal genes was increased, suggesting that Arc can control expression in the absence of activated signalling pathways. Taken together, these data establish Arc as a master regulator of neuronal activity-dependent gene expression and suggest that it plays a significant role in the pathophysiology of AD.
... Durch die anschließende Prozessierung von β -CTF durch die γ -Sekretase an der γ -Schnittstelle kommt es zur Sezernierung von pathogenem Aβ extrazellulär sowie durch den ε -Schnitt zur Bildung von genregulatorischem AICD (engl. APP intracellular domain) intrazellulär[56,229] (Abb.1). Analog zu NICD scheint auch AICD die Transkription von zahlreichen Genen zu regulieren. ...
... MEF WT wurden als Kontrolle verwendet. Alle Angaben sind als [%] -Anteil von MEF WT zu sehen.Diese Ergebnisse deuten darauf hin, dass ICD -Moleküle von APP und APLP2 einen Einfluss auf die 2+ -Homöostase ausüben, denn sowohl ICD -defiziente Zellen als auch die Zelllinie mit einem funktionslosen AICD zeigen signifikante Veränderungen gegenüber der Kontrolle MEF WT.Fe65 ist ein Bestandteil vom sogenannten AFT -Komplex und soll an der Translokation von AICD in denNukleus beteiligt sein, wo AICD mit Tip60 eine Verbindung eingeht und genregulatorische Eigenschaften entfaltet[56]. Aufgrund der kontroversen Literaturlage war es an dieser Stelle wichtig, den Einfluss von Fe65 genauer zu differenzieren. ...
... Similar to NICD, AICD has also been identified as a transcription factor, which forms a ternary complex with Fe65 and Tip60 (Borg Y. Cho, H.-G. Bae, E. Okun et al. Pharmacology & Therapeutics 235 (2022) 108122 et al., 1996Cao & Sudhof, 2001;Gao & Pimplikar, 2001;Pardossi-Piquard & Checler, 2012). NICD interacts with the transcription factor CSL and enters the nucleus to modulate the transcription of target genes (Baik et al., 2015;Bray, 2006). ...
... Likewise, AICD is stabilized by interaction with Fe65 and translocates to the nucleus, where the AICD-Fe65 complex associates with a histone acetyltransferase Tip60. The AICD-Fe65-Tip60 complex regulates the transcriptional activity; furthermore, some putative target genes of AICD have been proposed (Cao & Sudhof, 2001;Kimberly, Zheng, Guenette, & Selkoe, 2001). As a transcription factor, the AICD-Fe65-Tip60 complex regulates the transcription of diverse genes (Jo et al., 2010). ...
Amyloid precursor protein (APP) is an evolutionarily conserved transmembrane protein and a well-characterized precursor protein of amyloid-beta (Aβ) peptides, which accumulate in the brains of individuals with Alzheimer’s disease (AD)-related pathologies. Aβ has been extensively investigated since the amyloid hypothesis in AD was proposed. Besides Aβ, previous studies on APP and its proteolytic cleavage products have suggested their diverse pathological and physiological functions. However, their roles still have not been thoroughly understood. In this review, we extensively discuss the evolutionarily-conserved biology of APP, including its structure and processing pathway, as well as recent findings on the physiological roles of APP and its fragments in the central nervous system and peripheral nervous system. We have also elaborated upon the current status of APP-targeted therapeutic approaches for AD treatment by discussing inhibitors of several proteases participating in APP processing, including α-, β-, and γ-secretases. Finally, we have highlighted the future perspectives pertaining to further research and the potential clinical role of APP.
... APP fragments containing the Cter domain can also exert synaptic functions. AICD released upon γsecretase cleavage of APP-CTFs can be transported into the nucleus where they can form a transcription complex with Fe65 and Tip60 which modulates the expression of certain genes, some of which being related to synaptic transmission 198 . Furthermore, some of the work performed in this thesis provides evidence that the regulation of certain synaptic markers such as synaptotagmin-7 are mediated by APP-CTFs after γ-secretase absence or inhibition 199 . ...
... These peptides play specific roles in synapses, implying that the regulation and quantity of these peptides might have direct consequences on the synaptic pathology. As an example, the AICD fragment has been shown to function as a transcription factor, which regulates gene expression198,476 . However, this was not the case for the Syt7 gene, in which mRNA levels were not changed under PSKO conditions and thus not caused by deficits of the AICD fragment. ...
Alzheimer’s disease (AD) is a progressive neurodegenerative disease which affects 47 million people worldwide, being the most prominent type of dementia. The etiology of the disease is unknown but genetic evidence from the familial form of the disease indicates that the amyloid precursor protein (APP) plays a key role in the pathology. Importantly, APP is the substrate in the proteolytic reaction producing Aβ peptides which compose the amyloid plaques, one of the main pathological hallmarks in AD brain. In addition, APP is ubiquitously expressed by neurons where it interacts with multiple presynaptic proteins but the role of these interactions is elusive.The aim of my thesis was to study the physiological and pathological functions of APP related to its location at the presynapse. First, we studied the consequences on presynaptic mechanisms of the genetic deletion of presenilin, the catalytic subunit of γ-secretase, the intramembrane protease which cleaves APP. We observed that in absence of presenilin, APP accumulates in axons. By combining optogenetic to electrophysiology, we assessed synaptic transmission and plasticity in the CA3 region of the hippocampus. The presynaptic facilitation, the increase in synaptic vesicle release during repetitive stimulation, was altered whereas the basal neurotransmission was not. The impairment of presynaptic mechanisms was due to the accumulation of APP Cter, which decreases the abundancy of synaptotagmin-7, a calcium sensor essential for facilitation. Using a similar approach, we investigated the consequences of the genetic deletion of APP itself and observed again an impairment of presynaptic facilitation. Together, these results demonstrate the importance of APP homeostasis in presynaptic plasticity.I then investigated possible alterations of APP, other than the amyloid peptides, in the AD brain. I discovered that APP dramatically accumulates together with presynaptic proteins around dense-core amyloid plaques in human AD brain. In addition, the Nter domain, but not the Cter domain of APP is enriched in the core of amyloid plaques uncovering a potential pathological role of the secreted APP Nter in dense-core plaques. Ultrastructural analysis of APP accumulations reveals abundant multivesicular bodies containing presynaptic vesicle proteins and autophagosomal built-up of APP. Finally, we observed that outside the APP accumulations, presynaptic proteins were downregulated, in the neuropil area of the outer molecular layer of the dentate gyrus. Altogether, the data I collected during my thesis supports a role of presynaptic APP in physiology and in AD pathology and highlights APP accumulations as a pathological site where presynaptic proteins are mis-distributed.
... The interactomes of AICD, both in phosphorylated and unphosphorylated conditions are well known [16,17], and their roles in transcriptional transactivation have been well studied [18,19]. Besides that, AICD targets key regulatory proteins affecting cellular physiology like GSK-3β, p53 and EGFR [20, 21, and 22]. ...
... This was unexpected because in the amyloid pathway AICD is stabilised implicating that it plays some role in AD pathophysiology [54]. This role of AICD till date is disputed, but our data establishes that AICD reverses the toxic effect of Aβ to a large extent-and this putative protective role of AICD (by differentially affecting the degradome) is in concordance with the studies which implicate AICD in neuronal development and plasticity [17,18,19]. Of the up regulated set, hsa-miR-221-3p, hsa-miR-222-3p and hsa-miR-155-5p are implicated in AD [55][56][57]. ...
Altered expressions of protein-coding genes and microRNAs have been implicated in the pathogenesis of Alzheimer's disease (AD). The disrupted miRNA landscape (degradome) has been studied in AD mainly from the Aβ perspective. That Amyloid Precursor Protein C- terminal Domain (AICD) is involved in altering the cellular transcriptome has been reported, but its role in the degradome is still unexplored. The involvement of other long non-coding RNAs (lncRNA) is being realized recently. Using small RNA sequencing from an AD cell model, we observed perturbations in the levels of 47 miRNAs deregulated between the control and Aβ groups, and 26 deregulated miRNAs between the control and AICD + Aβ (AD) groups. Additionally, using a novel bioinformatics pipeline, we obtained a total of 263 differentially expressed lncRNAs in Aβ versus control group, and 41 deregulated lncRNAs in the AD versus control group. Effect of Aβ and AICD, individually and in combination, were validated with top regulated miRNA hits. Several of these miRNAs were found to target key Receptor Tyrosine Kinase (RTK) - many of which are implicated in AD. Understanding the total cellular non-coding transcriptome in the context of AD is likely to open up new putative targets for the disease intervention.
... More importantly, membrane-tethering of the AICD fragment was sufficient to reproduce endolysosome defects in combined PSEN-and APP-deficient cells, questioning the existence of a direct nuclear signaling role for AICD, as proposed previously. [80][81][82][83][84] APP-mediated downstream signaling is largely regulated through its YNPTY motif, with critical mutations abolishing the effects of mAICD on endolysosomal homeostasis (Figures 4 and 7). Inhibiting Abl kinase through Imatinib significantly rescued endolysosomal defects in PSENdKO cells ( Figures S5E-S5H), supporting a direct contribution of Abl-mediated downstream signaling on LE/Lys. ...
... APP could also participate in synaptic plasticity through the function of AICD/AID, the intracellular peptide that is generated after γ-secretase cleavage. In more detail, it was shown that AICD/AID forms complexes with the multidomain adaptor protein Fe65 and the histone acetyltransferase Tip60, which is subsequently transferred to the nucleus where it acts as a transcription factor [47,48]. Importantly, it was shown in vitro that AICD/AID participates in phosphoinositide-mediated intracellular calcium (Ca 2+ ) signaling [49]. ...
After several years of research in the field of Alzheimer’s disease (AD), it is still unclear how amyloid-beta (Aβ) and Tau, two key hallmarks of the disease, mediate the neuropathogenic events that lead to AD. Current data challenge the “Amyloid Cascade Hypothesis” that has prevailed in the field of AD, stating that Aβ precedes and triggers Tau pathology that will eventually become the toxic entity in the progression of the disease. This perspective also led the field of therapeutic approaches towards the development of strategies that target Aβ or Tau. In the present review, we discuss recent literature regarding the neurotoxic role of both Aβ and Tau in AD, as well as their physiological function in the healthy brain. Consequently, we present studies suggesting that Aβ and Tau act independently of each other in mediating neurotoxicity in AD, thereafter, re-evaluating the “Amyloid Cascade Hypothesis” that places Tau pathology downstream of Aβ. More recent studies have confirmed that both Aβ and Tau could propagate the disease and induce synaptic and memory impairments via the amyloid precursor protein (APP). This finding is not only interesting from a mechanistic point of view since it provides better insights into the AD pathogenesis but also from a therapeutic point of view since it renders APP a common downstream effector for both Aβ and Tau. Subsequently, therapeutic strategies that act on APP might provide a more viable and physiologically relevant approach for targeting AD.
... Studies showing AICD-dependent activation of GSK3β or protein kinase A (PKA) all relied on analysis of whole-brain or whole-cell lysates [16][17][18]43,47]. In those studies, the impact of AICD was reported to depend on kinase activation as well as nuclear signaling, and modulation of NMDAR and L-type Ca 2+ channels, which are lacking in the axoplasm [15,16,[48][49][50][51]. Together, these data strongly indicate that not only the increased production but also the subcellular compartment where the AICD fragment is generated could affect its pathophysiological function. ...
The amyloid precursor protein (APP) is a key molecular component of Alzheimer’s disease (AD) pathogenesis. Proteolytic APP processing generates various cleavage products, including extracellular amyloid beta (Aβ) and the cytoplasmic APP intracellular domain (AICD). Although the role of AICD in the activation of kinase signaling pathways is well established in the context of full-length APP, little is known about intracellular effects of the AICD fragment, particularly within discrete neuronal compartments. Deficits in fast axonal transport (FAT) and axonopathy documented in AD-affected neurons prompted us to evaluate potential axon-autonomous effects of the AICD fragment for the first time. Vesicle motility assays using the isolated squid axoplasm preparation revealed inhibition of FAT by AICD. Biochemical experiments linked this effect to aberrant activation of selected axonal kinases and heightened phosphorylation of the anterograde motor protein conventional kinesin, consistent with precedents showing phosphorylation-dependent regulation of motors proteins powering FAT. Pharmacological inhibitors of these kinases alleviated the AICD inhibitory effect on FAT. Deletion experiments indicated this effect requires a sequence encompassing the NPTY motif in AICD and interacting axonal proteins containing a phosphotyrosine-binding domain. Collectively, these results provide a proof of principle for axon-specific effects of AICD, further suggesting a potential mechanistic framework linking alterations in APP processing, FAT deficits, and axonal pathology in AD.
... Recent findings suggest that APP and PSs are the center of a complex network of interactions with many different intracellular adaptors, but the role of these proteins in the physiology or pathology is still unknown [87]. APP contains a YENPTY motif that has been previously described as an internalization motif, which now has been recognized to be involved as a key player in the regulation of multiple interactions with intracellular proteins [88]. The significance of the motif, which is typical of the receptor (TKR) and non-receptor tyrosine kinases (TK), in amyloid formation and in general for AD development is under investigation. ...
The pore-forming subunits (α subunits) of voltage-gated sodium channels (VGSC) are encoded in humans by a family of nine highly conserved genes. Among them, SCN1A, SCN2A, SCN3A, and SCN8A are primarily expressed in the central nervous system. The encoded proteins Nav1.1, Nav1.2, Nav1.3, and Nav1.6, respectively, are important players in the initiation and propagation of action potentials and in turn of the neural network activity. In the context of neurological diseases, mutations in the genes encoding Nav1.1, 1.2, 1.3 and 1.6 are responsible for many forms of genetic epilepsy and for Nav1.1 also of hemiplegic migraine. Several pharmacological therapeutic approaches targeting these channels are used or are under study. Mutations of genes encoding VGSCs are also involved in autism and in different types of even severe intellectual disability (ID). It is conceivable that in these conditions their dysfunction could indirectly cause a certain level of neurodegenerative processes; however, so far, these mechanisms have not been deeply investigated. Conversely, VGSCs seem to have a modulatory role in the most common neurodegenerative diseases such as Alzheimer’s, where SCN8A expression has been shown to be negatively correlated with disease severity.
... The large extracellular domain of APP, known as secreted APP, has a variety of functions (Mattson et al., 1993;Furukawa et al., 1996;Mockett et al., 2017). The intracellular portion of APP (known as the APP intracellular domain), similar to the homologous portion of the Notch protein, translocates to the nucleus, although its function there is not well understood (Cao and Sudhof, 2001;Gao and Pimplikar, 2001). Finally, APP is a damage response protein. ...
The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the β-amyloid peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aβ are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase in App gene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. GCaMP8f Ca ²⁺ reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown of App block the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aβ – either fibrillar or oligomeric – has no effect. In culture, APP Swe (a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and βIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model – both females and males – neurons have elevated levels of APP protein that invade the AIS. In vivo as in vitro, this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD.
SIGNIFICANCE:
While the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity. In vivo and in vitro, modest amounts of excess APP alter the properties of the axon initial segment (AIS). The Aβ peptide derived from APP is without effect. Consistent with the observed changes in the AIS which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the AIS suggesting a relationship with AD dementia.
... The tripartite complex of the APP intracellular domain (AICD), the nuclear adaptor protein Fe65, and the HAT TIP60 provides a clue to the mechanism involved in aberrant histone acetylation in AD neurons. 185 An integrated multiomics approach has identified H3K9ac and H3K27ac as the most significant enrichment specific to AD. Increasing these modifications exacerbates AD-related neurodegeneration in the Drosophila AD model. 186 Exposure of cortical and hippocampal cultures to Aβ oligomers increased acetylation in H3K14, resulting in loss of dendritic spines, which was reverted by HAT inhibitors. ...
Our nervous system, especially the brain is arguably the most complex, specialized and also most finely tuned organ of our body, controlling major fundamental and advanced functions that make us human. Neurological disorders, caused by any type of malfunctioning of the nervous system, are major healthcare burden. Primary focus of the large groups of researchers had been on finding genetic and environmental factors behind such disorders. With our growing understanding in the field of epigenetics and a phenomenal progress in advanced technologies contributing to chromatin biology research, a paradigm shift has been observed in disease research toward elucidating the chromatin link of human disorders. In this chapter, we provide a detailed analysis of the recent progress and understanding on contribution of epigenetic players in human neurological disorders.
... The similarity between APP/APLPs and Notch processing (see below) led some authors to hypothesize that the APP and APLPs [50] Intracellular C-terminal Domain (AICD) could possess signaling functions (see [51,52] for a review). Reporter gene assays based on Gal4-fusion constructs suggested that AICD could act as transcriptional regulator [53,54], but the controversy about the transcriptional activity of AICD and its target genes has only grown since then [55,56]. ...
Most neurodegenerative diseases have the characteristics of protein folding disorders, i.e., they cause lesions to appear in vulnerable regions of the nervous system, corresponding to protein aggregates that progressively spread through the neuronal network as the symptoms progress. Alzheimer’s disease is one of these diseases. It is characterized by two types of lesions: neurofibrillary tangles (NFTs) composed of tau proteins and senile plaques, formed essentially of amyloid peptides (Aβ). A combination of factors ranging from genetic mutations to age-related changes in the cellular context converge in this disease to accelerate Aβ deposition. Over the last two decades, numerous studies have attempted to elucidate how structural determinants of its precursor (APP) modify Aβ production, and to understand the processes leading to the formation of different Aβ aggregates, e.g., fibrils and oligomers. The synthesis proposed in this review indicates that the same motifs can control APP function and Aβ production essentially by regulating membrane protein dimerization, and subsequently Aβ aggregation processes. The distinct properties of these motifs and the cellular context regulate the APP conformation to trigger the transition to the amyloid pathology. This concept is critical to better decipher the patterns switching APP protein conformation from physiological to pathological and improve our understanding of the mechanisms underpinning the formation of amyloid fibrils that devastate neuronal functions.
... Having shown that levels of AICD and Fe65 inversely correlated with levels of CHCHD6, we further examined whether AICD and Fe65 could directly bind the CHCHD6 promoter. The histone acetyltransferase Tip60 forms a complex with the cytoplasmic tail of APP and the nuclear adaptor protein Fe65, enhancing the binding capacity of the complex to target gene promoters [6]. We therefore overexpressed AICD and Fe65 together with Tip60 in HEK293 cells. ...
The mechanistic relationship between amyloid-beta precursor protein (APP) processing and mitochondrial dysfunction in Alzheimer’s disease (AD) has long eluded the field. Here, we report that coiled-coil-helix-coiled-coil-helix domain containing 6 (CHCHD6), a core protein of the mammalian mitochondrial contact site and cristae organizing system, mechanistically connects these AD features through a circular feedback loop that lowers CHCHD6 and raises APP processing. In cellular and animal AD models and human AD brains, the APP intracellular domain fragment inhibits CHCHD6 transcription by binding its promoter. CHCHD6 and APP bind and stabilize one another. Reduced CHCHD6 enhances APP accumulation on mitochondria-associated ER membranes and accelerates APP processing, and induces mitochondrial dysfunction and neuronal cholesterol accumulation, promoting amyloid pathology. Compensation for CHCHD6 loss in an AD mouse model reduces AD-associated neuropathology and cognitive impairment. Thus, CHCHD6 connects APP processing and mitochondrial dysfunction in AD. This provides a potential new therapeutic target for patients.
... Many studies have shown that iron irregularities are seen in the onset and progression of AD and this leads to high production of APP by causing loss of function in various enzymes that require iron as a cofactor (Duce et al., 2010;Peters et al., 2015;Chen et al., 2019). It is also stated that the transcription of APP in the nucleus is related to the activation of Tip60 (Kinoshita et al., 2002) and the small cytoplasmic domain of APP (APP-CT) interacts with Tip60 to mediate transactivation of a reporter gene (Cao & Südhof, 2001). However, there is no study in the literature on the role of Tip60 in iron metabolism. ...
Hepcidin (HAMP), an iron regulatory hormone synthesized by liver hepatocytes, works together with ferritin (FTH) and ferroportin (FPN) in regulating the storage, transport, and utilization of iron in the cell. Epigenetic mechanisms, especially acetylation, also play an important role in the regulation of iron metabolism. However, a target protein has not been mentioned yet. With this preliminary study, we investigated the effect of histone acetyltransferase TIP60 on the expression of HAMP, FTH, and FPN. In addition, how the depletion of Tip60, which regulates the circadian system, affects the daily expression of Hamp was examined at six Zeitgeber time (ZT) points. For this purpose, liver-specific Tip60 knockout mice (mutant) were produced with tamoxifen-inducible Cre/lox recombination and an iron overload model in mice was generated. While HAMP and FTH expressions decreased, FPN expression increased in the mutant group. Interestingly, there was no change in the iron content. A significant increase was observed in the expressions of HAMP, FTH, and FPN and total liver iron content in the liver tissue of the iron overload group. Since intracellular iron concentration is involved in regulating the circadian clock, temporal expression of Hamp was investigated in control and mutant groups at six ZT points. In the control group, Hamp accumulated in a circadian manner with maximal and minimal levels reaching around ZT16 and ZT8, respectively. In the mutant group, there was a significant reduction in Hamp expression in the light phase ZT0 and ZT4 and in the dark phase ZT16. These data are the first findings demonstrating a possible relationship between Tip60 and iron metabolism.
... While more genes are downregulated than upregulated in response to the loss of TIP60, the amplitude of change is generally larger in the upregulated genes, suggesting that we also observed gene expression changes secondary to cellular events such as cell cycle arrest. H2AZ acetylation has been correlated with active transcription, consistent with a role of TIP60 in H2AZ acetylation and gene activation, albeit less locus-specific than previously reported [82][83][84]. H2AZ is found broadly at the transcription start site (TSS) of active and inactive genes as well as in gene bodies of inactive genes [63]. In contrast, acetylated H2AZ is shown to exclusively accumulate at the TSS of active genes [63]. ...
Histone acetylation is essential for initiating and maintaining a permissive chromatin conformation and gene transcription. Dysregulation of histone acetylation can contribute to tumorigenesis and metastasis. Using inducible cre-recombinase and CRISPR/Cas9-mediated deletion, we investigated the roles of the histone lysine acetyltransferase TIP60 (KAT5/HTATIP) in human cells, mouse cells, and mouse embryos. We found that loss of TIP60 caused complete cell growth arrest. In the absence of TIP60, chromosomes failed to align in a metaphase plate during mitosis. In some TIP60 deleted cells, endoreplication occurred instead. In contrast, cell survival was not affected. Remarkably, the cell growth arrest caused by loss of TIP60 was independent of the tumor suppressors p53, INK4A and ARF. TIP60 was found to be essential for the acetylation of H2AZ, specifically at lysine 7. The mRNA levels of 6236 human and 8238 mouse genes, including many metabolism genes, were dependent on TIP60. Among the top 50 differentially expressed genes, over 90% were downregulated in cells lacking TIP60, supporting a role for TIP60 as a key co-activator of transcription. We propose a primary role of TIP60 in H2AZ lysine 7 acetylation and transcriptional activation, and that this fundamental role is essential for cell proliferation. Growth arrest independent of major tumor suppressors suggests TIP60 as a potential anti-cancer drug target.
... Furthermore, we found the amyloid beta precursor protein-binding family B member 1 (APBB1) that binds to the intracellular fragment of APP (APP_ICD). Both LRP1 and APBB1 act together in gene expression [109,110]. APBB1 also co-localizes with APP in the ER and/or Golgi and, presumably, in endosomes, where it regulates the APP metabolism [111]. Interestingly, the above-mentioned TAP prey and γ-secretase substrate LRP1 modulates APBB1/APP-mediated gene activation [112,113]. ...
The very large G-protein-coupled receptor 1 (VLGR1/ADGRV1) is the largest member of the adhesion G-protein-coupled receptor (ADGR) family. Mutations in VLGR1/ADGRV1 cause human Usher syndrome (USH), a form of hereditary deaf-blindness, and have been additionally linked to epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying the development of these diseases are still unknown. Our study aimed to identify novel, previously unknown protein networks associated with VLGR1 in order to describe new functional cellular modules of this receptor. Using affinity proteomics, we have identified numerous new potential binding partners and ligands of VLGR1. Tandem affinity purification hits were functionally grouped based on their Gene Ontology terms and associated with functional cellular modules indicative of functions of VLGR1 in transcriptional regulation, splicing, cell cycle regulation, ciliogenesis, cell adhesion, neuronal development, and retinal maintenance. In addition, we validated the identified protein interactions and pathways in vitro and in situ. Our data provided new insights into possible functions of VLGR1, related to the development of USH and epilepsy, and also suggest a possible role in the development of other neuronal diseases such as Alzheimer’s disease.
... The HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit) (1:10,000) were purchased from GE. The plasmids APP-Gal4, Gal4-UAS-luciferase (encoding firefly luciferase) were kindly provided from Dr. Thomas Südof, and have been described (Cao and Sudhof, 2001). The previously reported ubiquilin-1 expressing plasmid was constructed from the pCMV vector and was kindly provided by Dr. Mervyn J. Monteiro (Mah et al., 2000). ...
Alzheimer’s disease (AD) belongs to a class of diseases characterized by progressive accumulation and aggregation of pathogenic proteins, particularly Aβ proteins. Genetic analysis has identified UBQLN1 as an AD candidate gene. Ubiquilin-1 levels reduce with AD progression, suggesting a potential loss-of-function mechanism. The ubiquilin-1 protein is involved in protein quality control (PQC), which plays essential roles in cellular growth and normal cell function. Ubiquilin-1 regulates γ-secretase by increasing endoproteolysis of PS1, a key γ-secretase component. Presently, the effects of ubiquilin-1 on cellular physiology as well as Aβ-related events require further investigation. Here, we investigated the effects of ubiquilin-1 on cellular growth and viability in association with APP (amyloid-β protein precursor), APP processing-related β-secretase (BACE1, BACE) and γ-secretase using cell and animal-based models. We showed that loss-of-function in Drosophila ubqn suppresses human APP and human BACE phenotypes in wing veins and altered cell number and tissue compartment size in the wing. Additionally, we performed cell-based studies and showed that silencing UBQLN1 reduced cell viability and increased caspase-3 activity. Overexpression of UBQLN1 significantly reduced Aβ levels. Furthermore, pharmacological inhibition of γ-secretase increased ubiquilin-1 protein levels, suggesting a mechanism that regulates ubiquilin-1 levels which may associate with reduced Aβ reduction by inhibiting γ-secretase. Collectively, our results support not only a loss-of-function mechanism of ubiquilin-1 in association with AD, but also support the significance of targeting ubiquilin-1-mediated PQC as a potential therapeutic strategy for AD.
... Once in the nucleus, AICD can promote gene transcription (Cao, 2001). AICD may contribute to control of gene expression programs (Xinwei, 2001), such as cell signalling pathways, cytoskeletal changes (Müller et al., 2007) and negative feedback of APP and Aβ (Bukhari et al., 2017). ...
Amyloid precursor protein (APP) and its cleavage fragment Amyloid-β (Aβ) have fundamental roles in Alzheimer’s disease (AD). Genetic alterations that either increase the overall dosage of APP or alter its processing to favour the generation of longer, more aggregation prone Aβ species, are directly causative of the disease. People living with one copy of APP are asymptomatic and reducing APP has been shown to lower the relative production of aggregation-prone Aβ species in vitro. For these reasons, reducing APP expression is an attractive approach for AD treatment and prevention. In this review, we will describe the structure and the known functions of APP and go on to discuss the biological consequences of APP knockdown and knockout in model systems. We highlight progress in therapeutic strategies to reverse AD pathology via reducing APP expression. We conclude that new technologies that reduce the dosage of APP expression may allow disease modification and slow clinical progression, delaying or even preventing onset.
... For instance, the inclusion of a 6 nt microexon in mature amyloid-beta precursor protein binding, family b member 1 (APBB1) mRNA is modulated during differ-entiation of mouse embryonic stem cells (mESCs) into cortical glutamatergic neurons and strongly affects the properties of the cognate protein [5]. APBB1 protein comprises two phosphotyrosine-binding domains named PTB1 and PTB2, which mediate the interaction with the histone acetyltransferase Kat5/Tip60 and the amyloid-beta precursor protein (APP), respectively [127]. Interestingly, the inclusion of the microexon in the APBB1 mRNA adds two charged residues in the PTB1 domain of the corresponding protein and significantly enhances its interaction with the histone acetyltransferase Kat5/Tip60, and to a lesser extent, with APP [5]. ...
The advance of experimental and computational techniques has allowed us to highlight the existence of numerous different mechanisms of RNA maturation, which have been so far unknown. Besides canonical splicing, consisting of the removal of introns from pre-mRNA molecules, non-canonical splicing events may occur to further increase the regulatory and coding potential of the human genome. Among these, splicing of microexons, recursive splicing and biogenesis of circular and chimeric RNAs through back-splicing and trans-splicing processes, respectively, all contribute to expanding the repertoire of RNA transcripts with newly acquired regulatory functions. Interestingly, these non-canonical splicing events seem to occur more frequently in the central nervous system, affecting neuronal development and differentiation programs with important implications on brain physiology. Coherently, dysregulation of non-canonical RNA processing events is associated with brain disorders, including brain tumours. Herein, we summarize the current knowledge on molecular and regulatory mechanisms underlying canonical and non-canonical splicing events with particular emphasis on cis-acting elements and trans-acting factors that all together orchestrate splicing catalysis reactions and decisions. Lastly, we review the impact of non-canonical splicing on brain physiology and pathology and how unconventional splicing mechanisms may be targeted or exploited for novel therapeutic strategies in cancer.
... A consensus has been reached, suggesting that the internal C-terminal domain of the amyloid precursor protein or AICD, in interaction with Fe65, is translocated to the cell nucleus [13]. In this cell compartment, it would form a transcriptively active complex [14] with the histone acetyl transferase Tip60 [15,16]. This complex would be recruited at DSB sites [17]. ...
Recently, we showed that DNA double-strand breaks (DSBs) are increased by the Aβ42-amyloid peptide and decreased by all-trans retinoic acid (RA) in SH-SY5Y cells and C57BL/6J mice. The present work was aimed at investigating DSBs in cells and murine models of Alzheimer’s disease carrying the preseniline-1 (PS1) P117L mutation. We observed that DSBs could hardly decrease following RA treatment in the mutated cells compared to the wild-type cells. The activation of the amyloidogenic pathway is proposed in the former case as Aβ42- and RA-dependent DSBs changes were reproduced by an α-secretase and a γ-secretase inhibitions, respectively. Unexpectedly, the PS1 P117L cells showed lower DSB levels than the controls. As the DSB repair proteins Tip60 and Fe65 were less expressed in the mutated cell nuclei, they do not appear to contribute to this difference. On the contrary, full-length BRCA1 and BARD1 proteins were significantly increased in the chromatin compartment of the mutated cells, suggesting that they decrease DSBs in the pathological situation. These Western blot data were corroborated by in situ proximity ligation assays: the numbers of BRCA1-BARD1, not of Fe65-Tip60 heterodimers, were increased only in the mutated cell nuclei. RA also enhanced the expression of BARD1 and of the 90 kDa BRCA1 isoform. The increased BRCA1 expression in the mutated cells can be related to the enhanced difficulty to inhibit this pathway by BRCA1 siRNA in these cells. Overall, our study suggests that at earlier stages of the disease, similarly to PS1 P117L cells, a compensatory mechanism exists that decreases DSB levels via an activation of the BRCA1/BARD1 pathway. This supports the importance of this pathway in neuroprotection against Alzheimer’s disease.
... En effet, grâce à sa séquence spécifique Y 682 ENPTY 687 , un motif d'endocytose médiée par la clathrine, l'AICD interagit avec Fe65 afin d'effectuer une translocation vers le noyau (Konietzko et al., 2010). L'AICD exerce ainsi une toxicité via des mécanismes dépendants de la transcription en formant un complexe tripartite avec Fe65 et l'histone acétyltransférase Tip60 dans le noyau (Cao and Südhof, 2001), induisant l'expression de GSK-3β, suivie d'une phosphorylation de la protéine Tau, qui contribuerait à la pathogenèse de la MA (Kim et al., 2003). La génération d'AICD a lieu au niveau de la membrane plasmique et/ou dans les endosomes précoces (Vingtdeux et al., 2007a). ...
Dans le cerveau des malades atteints de la maladie d’Alzheimer (MA), deux types de lésions coexistent : les dépôts parenchymateux formés d’agrégats de peptides amyloïdes (A) et l’agrégation intra-neuronale de protéines Tau anormalement et hyperphosphorylées. Les peptides Aβ sont issus du clivage séquentiel de la protéine précurseur du peptide amyloïde (APP) par deux enzymes, la β-secrétase et la γ-secrétase. En grande majorité, les molécules actuellement en essais cliniques ciblent l’une ou l’autre des deux pathologies, or la MA se définit par la co-existence de ces deux lésions. Ainsi, plusieurs études suggèrent un lien synergique entre les deux processus lésionnels, et un traitement qui agirait à la fois sur la pathologie amyloïde et sur la pathologie Tau apparaîtrait alors comme une stratégie thérapeutique de grand intérêt. Au carrefour de ces deux pathologies se trouve la régulation de l’homéostasie protéique faisant appel notamment au système endosome/lysosome. Plusieurs familles de molécules dérivées de la chloroquine issues du laboratoire agissent sur ce système endosome / lysosome essentiel au métabolisme de l’APP et au métabolisme de la protéine Tau. Une étude de la relation structure-activité de ces familles de molécules a été réalisée, et deux molécules, nommées MAGS et PEL, qui partagent un effet modulateur de la β-secrétase associé ou non à une activité lysosomotropique in vitro ont été identifiées. En termes de structure chimique, MAGS et PEL diffèrent uniquement par un atome d'azote. Ces molécules ont montré une capacité in vitro à bloquer la production de peptides Aβ. Identifier laquelle parmi plusieurs activités pharmacologiques in vitro est nécessaire pour obtenir l’efficacité désirée in vivo est essentiel pour la poursuite du développement de médicaments contre les processus physiopathologiques de la MA. Premièrement, ce travail a permis de démontrer que l’activité lysosomotropique n’était pas essentielle à l’efficacité de nos molécules in vivo, et qu’un traitement curatif avec la molécule PEL permettait de restaurer les fonctions cognitives, de diminuer l’hyperphosphorylation de Tau et la dégénérescence neurofibrillaire dans un modèle murin de pathologie Tau hippocampique. Deuxièmement, la molécule PEL a également permis une restauration complète de la mémoire à court et long-terme ainsi que la diminution de l’agrégation pathologique du peptide Aβ en plaques amyloïdes dans un modèle murin d’amyloïdogenèse. Enfin, nous avons pu mettre en évidence plusieurs voies sur lesquelles la molécule PEL pourrait agir, notamment la composante inflammatoire et en particulier l’astrogliose dont la diminution significative a été constatée dans les deux modèles murins étudiés, ainsi que la protéine PP2AC, principale phosphatase de Tau, dont la synthèse a été augmentée consécutivement au traitement avec PEL. Une augmentation de la protéine régulatrice du trafic endosomal de la β-secrétase, GGA1, a également été observée. Ce travail apporte des éléments nouveaux pour la compréhension de l’activité de nos molécules et suggère qu’au-delà de la modulation de l’activité -secrétase, PEL possède une action sur l’astrogliose et la réponse inflammatoire.
... Next, we compared the nuclear import kinetics of GFP and β 1ICD-GFP. Other cleaved ICDs, such as APP, are trafficked to the nucleus as part of a heteromeric complex (57). Such a complex is expected to have slower import kinetics than soluble GFP, which can diffuse directly through nuclear pores. ...
The voltage-gated Na+ channel β1 subunit, encoded by SCN1B, regulates cell surface expression and gating of α subunits, and participates in cell adhesion. β1 is cleaved by α/β and γ-secretases, releasing an extracellular domain and intracellular domain (ICD) respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on β1 function in breast cancer cells. Using a series of GFP-tagged β1 constructs, we show that β1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal β1-GFP levels occurred following γ-secretase inhibition, implicating endosomes, and/or the preceding plasma membrane, as important sites for secretase processing. Using live-cell imaging, we report β1-ICD-GFP accumulation in the nucleus. Furthermore, β1-GFP and β1ICD-GFP both increased Na+ current, whereas β1STOP-GFP, which lacks the ICD, did not, thus highlighting that the β1-ICD was necessary and sufficient to increase Na+ current measured at the plasma membrane. Importantly, although the endogenous Na+ current expressed in MDA-MB-231 cells is TTX-resistant (carried by Nav1.5), the Na+ current increased by β1-GFP or β1ICD-GFP was TTX-sensitive. Taken together, this work suggests that the β1-ICD is a critical regulator of β subunit function. Our data further support the notion that γ-secretase may play a key role in regulating β1 function in breast cancer cells. This work thus highlights proteolytic processing of β1 by secretase cleavage to be a relevant mechanism in diseases associated with abnormal β1 function.
... Furthermore, the Aβ 25-35 peptide promotes Ca 2+ influx by activating L-and T-type Ca 2+ channels in rat hippocampal slices (Li et al., 2010). The APP intracellular domain (AICD), a APP cleavage fragment, may act as a transcription factor to activate the Ca 2+ signaling system (Cao and Südhof, 2001;Leissring et al., 2002). Because of the self-aggregating characteristics of Aβ, Aβ oligomers can promote Ca 2+ influx through N-methyl-D-aspartic acid receptor (NMDAR) channels in a short period of time (Kelly and Ferreira, 2006). ...
Alzheimer’s disease (AD) is a neurodegenerative disease that is characterized by the production and deposition of β-amyloid protein (Aβ) and hyperphosphorylated tau, leading to the formation of β-amyloid plaques (APs) and neurofibrillary tangles (NFTs). Although calcium ions (Ca ²⁺ ) promote the formation of APs and NFTs, no systematic review of the mechanisms by which Ca ²⁺ affects the development and progression of AD has been published. Therefore, the current review aimed to fill the gaps between elevated Ca ²⁺ levels and the pathogenesis of AD. Specifically, we mainly focus on the molecular mechanisms by which Ca ²⁺ affects the neuronal networks of neuroinflammation, neuronal injury, neurogenesis, neurotoxicity, neuroprotection, and autophagy. Furthermore, the roles of Ca ²⁺ transporters located in the cell membrane, endoplasmic reticulum (ER), mitochondria and lysosome in mediating the effects of Ca ²⁺ on activating neuronal networks that ultimately contribute to the development and progression of AD are discussed. Finally, the drug candidates derived from herbs used as food or seasoning in Chinese daily life are summarized to provide a theoretical basis for improving the clinical treatment of AD.
... However, APP immunoreactivity increased by 47 ± 16% in treated hippocampal neurons (Fig. 5c). Considering that Fe65 translocates to the nucleus to activate the transcription of key genes [40], we decided to study its distribution between the nucleus (using DAPI staining) and the cytoplasm (using MAP1B immunoreactivity, Fig. 5d). As shown in the graph of Fig. 5e, the N/C ratio of Fe65 immunoreactivity decreased 28 ± 4% after chronic Aβo exposure compared to the control condition. ...
Amyloid beta peptide (Aβ) is tightly associated with the physiopathology of Alzheimer’s Disease (AD) as one of the most important factors in the evolution of the pathology. In this context, we previously reported that Aβ increases the expression of ionotropic purinergic receptor 2 (P2×2R). However, its role on the cellular and molecular Aβ toxicity is unknown, especially in human brain of AD patients. Using cellular and molecular approaches in hippocampal neurons, PC12 cells, and human brain samples of patients with AD, we evaluated the participation of P2×2R in the physiopathology of AD. Here, we reported that Aβ oligomers (Aβo) increased P2×2 levels in mice hippocampal neurons, and that this receptor increases at late Braak stages of AD patients. Aβo also increases the colocalization of APP with Rab5, an early endosomes marker, and decreased the nuclear/cytoplasmic ratio of Fe65 and PGC-1α immunoreactivity. The overexpression in PC12 cells of P2×2a, but not P2×2b, replicated these changes in Fe65 and PGC-1α; however, both overexpressed isoforms increased levels of Aβ. Taken together, these data suggest that P2×2 is upregulated in AD and it could be a key potentiator of the physiopathology of Aβ. Our results point to a possible participation in a toxic cycle that increases Aβ production, Ca²⁺ overload, and a decrease of PGC-1α. These novel findings put the P2×2R as a key novel pharmacological target to develop new therapeutic strategies to treat Alzheimer’s Disease.
... A possible explanation for this effect may involve the reversed regulation of APP695 and InsP3R genes at the mRNA and protein levels during differentiation [58]. The APP intracellular domain (AICD), which is released after InsP3R cleavage of APP may act as a transcription factor to activate the Ca 2+ signaling system [59,60]. As the cleavage fragments of APP are produced by different secretases, PSEN2 mutation has shown its effects on impairing the fusion between autophagosomes and lysosomes in PSEN2 T122R mutated SH-SY5Y cells [61]. ...
Alzheimer’s disease (AD) is a neurodegenerative disease with a high incidence rate. The main pathological features of AD are β-amyloid plaques (APs), which are formed by β-amyloid protein (Aβ) deposition, and neurofibrillary tangles (NFTs), which are formed by the excessive phosphorylation of the tau protein. Although a series of studies have shown that the accumulation of metal ions, including calcium ions (Ca2+), can promote the formation of APs and NFTs, there is no systematic review of the mechanisms by which Ca2+ affects the development and progression of AD. In view of this, the current review summarizes the mechanisms by which Ca2+ is transported into and out of cells and organelles, such as the cell, endoplasmic reticulum, mitochondrial and lysosomal membranes to affect the balance of intracellular Ca2+ levels. In addition, dyshomeostasis of Ca2+ plays an important role in modulating the pathogenesis of AD by influencing the production and aggregation of Aβ peptides and tau protein phosphorylation and the ways that disrupting the metabolic balance of Ca2+ can affect the learning ability and memory of people with AD. In addition, the effects of these mechanisms on the synaptic plasticity are also discussed. Finally, the molecular network through which Ca2+ regulates the pathogenesis of AD is introduced, providing a theoretical basis for improving the clinical treatment of AD.
Mounting evidence indicates that a physiological function of amyloid-β (Aβ) is to mediate neural activity-dependent homeostatic and competitive synaptic plasticity in the brain. I have previously summarized the lines of evidence supporting this hypothesis and highlighted the similarities between Aβ and anti-microbial peptides in mediating cell/synapse competition. In cell competition, anti-microbial peptides deploy a multitude of mechanisms to ensure both self-protection and competitor elimination. Here I review recent studies showing that similar mechanisms are at play in Aβ-mediated synapse competition and perturbations in these mechanisms underpin Alzheimer’s disease (AD). Specifically, I discuss evidence that Aβ and ApoE, two crucial players in AD, co-operate in the regulation of synapse competition. Glial ApoE promotes self-protection by increasing the production of trophic monomeric Aβ and inhibiting its assembly into toxic oligomers. Conversely, Aβ oligomers, once assembled, promote the elimination of competitor synapses via direct toxic activity and amplification of “eat-me” signals promoting the elimination of weak synapses. I further summarize evidence that neuronal ApoE may be part of a gene regulatory network that normally promotes competitive plasticity, explaining the selective vulnerability of ApoE expressing neurons in AD brains. Lastly, I discuss evidence that sleep may be key to Aβ-orchestrated plasticity, in which sleep is not only induced by Aβ but is also required for Aβ-mediated plasticity, underlining the link between sleep and AD. Together, these results strongly argue that AD is a disease of competitive synaptic plasticity gone awry, a novel perspective that may promote AD research.
We provide evidence here from loss-of-function studies in mice and gain-of-function studies in human cells (1, 2) which demonstrate control of APBB2 by the NLR (nucleotide-binding oligomerization domain-like receptor) family protein NOD2.
Down syndrome (DS) is caused by triplication of human chromosome 21 (HSA21). Although several HSA21 genes have been found to be responsible for aspects of DS, whether and how HSA21 genes interact with each other is poorly understood. DS patients and animal models present with a number of neurological changes, including aberrant connectivity and neuronal morphology. Previous studies have indicated that amyloid precursor protein (APP) and Down syndrome cell adhesion molecule (DSCAM) regulate neuronal morphology and contribute to neuronal aberrations in DS. Here, we report the functional interaction between the Drosophila homologs of these two genes, Amyloid precursor protein-like (Appl) and Dscam (Dscam1). We show that Appl requires Dscam to promote axon terminal growth in sensory neurons. Moreover, Appl increases Dscam protein expression post-transcriptionally. We further demonstrate that regulation of Dscam by Appl does not require the Appl intracellular domain or second extracellular domain. This study presents an example of functional interactions between HSA21 genes, providing insights into the pathogenesis of neuronal aberrations in DS.
Presenilin mutations that alter γ-secretase activity cause familial Alzheimer's disease (AD), whereas ApoE4, an apolipoprotein for cholesterol transport, predisposes to sporadic AD. Both sporadic and familial AD feature synaptic dysfunction. Whether γ-secretase is involved in cholesterol metabolism and whether such involvement impacts synaptic function remains unknown. Here, we show that in human neurons, chronic pharmacological or genetic suppression of γ-secretase increases synapse numbers but decreases synaptic transmission by lowering the presynaptic release probability without altering dendritic or axonal arborizations. In search of a mechanism underlying these synaptic impairments, we discovered that chronic γ-secretase suppression robustly decreases cholesterol levels in neurons but not in glia, which in turn stimulates neuron-specific cholesterol-synthesis gene expression. Suppression of cholesterol levels by HMG-CoA reductase inhibitors (statins) impaired synaptic function similar to γ-secretase inhibition. Thus, γ-secretase enables synaptic function by maintaining cholesterol levels, whereas the chronic suppression of γ-secretase impairs synapses by lowering cholesterol levels.
N‐methyl‐D‐aspartate receptors (NMDARs) are critical for the maturation and plasticity of glutamatergic synapses. In the hippocampus, NMDARs mainly contain GluN2A and/or GluN2B regulatory subunits. The amyloid precursor protein (APP) has emerged as a putative regulator of NMDARs, but the impact of this interaction to their function is largely unknown. By combining patch‐clamp electrophysiology and molecular approaches, we unravel a dual mechanism by which APP controls GluN2B‐NMDARs, depending on the life stage. We show that APP is highly abundant specifically at the postnatal postsynapse. It interacts with GluN2B‐NMDARs, controlling its synaptic content and mediated currents, both in infant mice and primary neuronal cultures. Upon aging, the APP amyloidogenic‐derived C‐terminal fragments, rather than APP full‐length, contribute to aberrant GluN2B‐NMDAR currents. Accordingly, we found that the APP processing is increased upon aging, both in mice and human brain. Interfering with stability or production of the APP intracellular domain normalized the GluN2B‐NMDARs currents. While the first mechanism might be essential for synaptic maturation during development, the latter could contribute to age‐related synaptic impairments. Rajão‐Saraiva et al. identified the amyloid precursor protein (APP) as an important regulator of NMDA receptors, acting through a dual age‐dependent mechanism. During postnatal development, APP controls GluN2B‐NMDAR synaptic content and currents, potentially contributing to synaptic maturation. Upon aging, the APP amyloidogenic intracellular fragments contribute to aberrant GluN2B‐NMDAR currents. This work highlights the importance of keeping APP processing under tight control, to ensure the normal functioning of glutamatergic synapses, being particularly relevant to understand age‐related synaptic impairments and Alzheimer's disease.
Inflammatory bowel disease (IBD) is an immune-mediated chronic intestinal disorder with major phenotypes; ulcerative colitis (UC), and Crohn's disease (CD). Multiple studies have identified over 240 IBD susceptibility loci. However, most studies have centered on European (EUR) and East Asian (EAS) populations. The prevalence of IBD in non-EUR, including African Americans (AAs), has risen in recent years. Here we present the first attempt to identify loci in AAs using a trans-ancestry Bayesian approach (MANTRA) accounting for heterogeneity between diverse ancestries while allowing for the similarity between closely related populations. We meta-analyzed GWAS and Immunochip data from a 2015 EUR meta-analysis of 38 155 IBD cases and 48 485 controls and EAS Immunochip study of 2824 IBD cases and 3719 controls, and our recent AA IBD GWAS of 2345 cases and 5002 controls. Across the major IBD phenotypes, we found significant evidence for 92% of the 2015 loci but also 3 IBD loci only established in latter studies. We detected 20 novel loci, all containing immunity-related genes or genes with other evidence for IBD or immune-mediated disease relevance: PLEKHG5;TNFSFR25 (encoding death receptor 3, receptor for TNFSF15 gene product TL1A), XKR6, ELMO1, BC021024;PI4KB;PSMD4, and APLP1 for IBD; AUTS2, XKR6, OSER1, TET2;AK094561, BCAP29, and APLP1 for CD; and GABBR1;MOG, DQ570892, SPDEF;ILRUN, SMARCE1;CCR7;KRT222;KRT24;KRT25, ANKS1A;TCP11, IL7, LRRC18;WDFY4, XKR6 and TNFSF4 for UC. Our study highlights the value of combining low-powered genomic studies from understudied populations of diverse ancestral backgrounds together with a high-powered study to enable novel locus discovery, including potentially important therapeutic IBD gene targets.
MUNC18-1/Syntaxin binding protein-1 (Stxbp1), binds Syntaxin-1. Together, these proteins regulate synaptic vesicle exocytosis and have a separate role in neuronal viability. In MUNC18-1/Stxbp1 null mutant neurons, Syntaxin-1 protein levels are 70% reduced. Here, we show that Dynamin-1 protein levels are reduced at least to the same extent, and DNM1 transcript levels are 50% reduced in MUNC18-1/Stxbp1 null mutant brain. Several, but not all, other endocytic proteins were also reduced, but to a lesser extent. The reduced Dynamin-1 expression was not observed in SNAP25 and MUNC13-1/2 null mutants, in which synaptic vesicle exocytosis is also blocked. Co-immunoprecipitation experiments demonstrated that Dynamin-1 and MUNC18-1 do not bind directly. Furthermore, MUNC18-1 levels were unaltered in neurons lacking all three Dynamin paralogs. Finally, overexpression of Dynamin-1 was not sufficient to rescue neuronal viability in MUNC18-1/Stxbp1 null mutant neurons, thus the reduction in Dynamin-1 is not the single cause for neurodegeneration of these neurons. The reduction of Dynamin-1 protein and mRNA, and other endocytosis proteins in Stxbp1 null mutant neurons suggests that MUNC18-1 directly or indirectly controls protein and mRNA levels of other presynaptic genes.
The formation and deposition of the-amyloid peptide (A) are largely thought to be responsible for the development of Alzheimer's disease (AD). Researchers have been perplexed by the sluggish to nonexistent association between the degree of clinical dementia and the amount of neuritic plaque disease in the human brain for a long time. This question has been resolved thanks to recent developments in our knowledge of the origins of amyloid disease. The solubility of A and the amount of A in various pools may now be more directly tied to disease state, according to substantial evidence. The make-up of these pools of A reflects various populations of amyloid deposits and is clearly correlated with the patient's clinical condition. Using imaging methods, such as novel amyloid imaging agents based on the chemical makeup of histology dyes, it is now possible to monitor the development of the disease in a living patient while also monitoring the amyloid pathology. It's interesting that these methods show that the A deposited in AD differs from that discovered in animal models. In general, deposited A does not exhibit the same physical and biochemical properties as the amyloid observed in AD and is more readily removed from the brain in animal studies. Important questions about the creation and evaluation of potential medicinal agents are brought up by this.
Tau aggregation in neurofibrillary tangles (NFTs) is closely associated with neurodegeneration and cognitive decline in Alzheimer’s disease (AD). However, the molecular signatures that distinguish between aggregation-prone and aggregation-resistant cell states are unknown. We developed methods for the high-throughput isolation and transcriptome profiling of single somas with NFTs from the human AD brain, quantified the susceptibility of 20 neocortical subtypes for NFT formation and death, and identified both shared and cell-type-specific signatures. NFT-bearing neurons shared a marked upregulation of synaptic transmission-related genes, including a core set of 63 genes enriched for synaptic vesicle cycling. Oxidative phosphorylation and mitochondrial dysfunction were highly cell-type dependent. Apoptosis was only modestly enriched, and the susceptibilities of NFT-bearing and NFT-free neurons for death were highly similar. Our analysis suggests that NFTs represent cell-type-specific responses to stress and synaptic dysfunction. We provide a resource for biomarker discovery and the investigation of tau-dependent and tau-independent mechanisms of neurodegeneration.
Human APOER2 is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail. APOER2-ECD contains several ligand binding domains (LBD) that are organized into exons with aligning phase junctions, which allows for in-frame exon cassette splicing events. We have identified 25 human APOER2 isoforms from cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events within the N-terminal LBD regions compared to 6 identified in the heart. APOER2 undergoes proteolytic cleavage in response to ligand binding that releases a C-terminal fragment (CTF) and transcriptionally active intracellular domain (ICD). We tested whether the diversity of human brain-specific APOER2 variants affects APOER2 cleavage. We found isoforms with differing numbers of ligand binding repeats generated different amounts of CTFs compared to full-length APOER2 (APOER2-FL). Specifically, APOER2 isoforms lacking exons 5-8 (Δex5-8) and lacking exons 4-6 (Δex4-6) generated the highest and lowest amounts of CTF generation, respectively in response to APOE peptide compared to APOER2-FL. The differential CTF generation of Δex5-8 and Δex4-6 coincides with the proteolytic release of the ICD which mediates transcriptional activation facilitated by the Mint1 adaptor protein. Functionally, we demonstrated loss of mouse Apoer2 decreased miniature event frequency in excitatory synapses which may be due to a decrease in total number of synapses and/or VAMP2 positive neurons. Lentiviral infection with human APOER2-FL or Δex4-6 isoform in Apoer2 knockout neurons restored the miniature event frequency, but not Δex5-8 isoform. These results suggest that human APOER2 isoforms have differential cleavage events and synaptic properties.SignificanceHumans and mice share virtually the same number of protein-coding genes. However, humans have greater complexity of any higher eukaryotic organisms by encoding multiple protein forms through alternative splicing modifications. Alternative splicing allows pre-mRNAs transcribed from genes to be spliced in different arrangements, producing structurally and functionally distinct protein variants that increases proteomic diversity and particularly prevalent in the human brain. Here, we identified 25 distinct human APOER2 splice variants from the cerebral cortex using gene-specific APOER2 primers where the majority are exon-skipping events that exclude N-terminal ligand binding regions of APOER2. We show that some of the APOER2 variants have differential proteolytic properties in response to APOE ligand and exhibit distinct synaptic properties.
Alzheimer’s Disease (AD), a common type of dementia, characterized by the presence of aggregated extracellular amyloid-beta (Aβ), intracellular hyper phosphorylation of tau protein and neurodegenerative with cognitive decline. It is projected that 141 million people will be suffering with AD by 2050 but no effective drug treatment is discovered without side effects. There is an urgent need for the application of alternative and non-pharmacological interventions for AD. Sporadically found that exercise or diet therapy or social activity may positively influence the AD. In this review we discussed the process of how Exercise-Eating pattern and Social inclusion (EES) has been shown to have fewer side effects and better adherence with AD. In this mechanism the EES can modulate the brain metabolic factors, brain-derived neurotrophic, ketone bodies, lactate, cathepsin-B, irisin, hormonal balance in AD. This review also described the potential biological mechanisms underlying exercise (modulation of biomolecule turnover, antioxidant and anti inflammation), eating pattern (bioactive compounds) and social inclusion that is very important to ameliorate the pathophysiological hallmarks of Alzheimer’s disease. Thus, this EES can be an effective approach to manage the neurodegenerative disorder as well as Alzheimer’s disease.
Amyloid precursor protein (APP) is associated with both familial and sporadic forms of Alzheimer’s disease. APP has two homologs, amyloid precursor-like protein 1 and 2 (APLP1 and APLP2), and they have functional redundancy. APP intracellular c-terminal domain (AICD), produced by sequential α- or β- and γ-secretase cleavages, is thought to control gene expression, similarly as the ICD of Notch. To investigate the role of APP family in transcriptional regulation, we examined gene expression changes in the cerebral cortex of APP/APLP1/APLP2 conditional triple knockout (cTKO) mice, in which APP family members are selectively inactivated in excitatory neurons of the postnatal forebrain. Of the 12 previously reported AICD target genes, only Nep and Npas4 mRNA levels were significantly reduced in the cerebral cortex of cTKO mice, compared to littermate controls. We further examined global transcriptional changes by RNA-seq and identified 189 and 274 differentially expressed genes in the neocortex and hippocampus, respectively, of cTKO mice relative to controls. Gene Ontology analysis indicated that these genes are involved in a variety of cellular functions, including extracellular organization, learning and memory, and ion channels. Thus, inactivation of APP family alters transcriptional profiles of the cerebral cortex and affects wide-ranging molecular pathways.
Alzheimer’s disease (AD) is a progressive neurodegeneration in vulnerable regions of the hippocampus and cerebral cortex that leads to loss in thinking abilities and dementia. The main pathological features of AD include loss of neurons and synapses, extracellular amyloid plaques composed primarily of amyloid β peptide 1–42, and intracellular neurofibrillary tangles which are mostly abnormally paired helical filaments of hyperphosphorylated tau protein. The precise mechanisms of neuronal cell deaths in AD are yet to be established. The amyloid hypothesis of AD states that the aberrant processing of the β-amyloid precursor protein (APP) and aggregation of amyloid β-peptide 1-42 (Aβ 1-42) plays a central role in neurodegeneration in AD. Normally, APP is cleaved by α-secretase to release an extracellular fragment known as sAPPα. In contrast, Aβ is produced when APP is cleaved by β-secretase (BACE1) and gamma-secretase. Cleavage of APP by β-secretase generates a soluble NH2-terminal fragment (sAPPβ) and a membrane-bound COOH-terminal fragment C99. Cleavage of C99 by gamma-secretase produces Aβ and an APP intracellular domain (AICD). It is generally believed that Aβ 1–42 is neurotoxic while sAPPα confers neuroprotection. Mutations in familial Alzheimer’s disease (FAD) genes, including those in APP and presenilins (which include presenilin-1, PS-1, and presenilin-2, PS-2), are associated with early-onset AD cases. Par-4 (prostate apoptosis response-4) is a cell death-promoting protein that was initially isolated as an apoptosis-associated protein by differential screening for genes upregulated in prostate cancer cells undergoing apoptosis. Par-4 is expressed in neurons, and it is found in both cytoplasmic and nucleus compartments. In this chapter, we provide emerging evidence that Par-4 is involved in neuronal cell death in a variety of neurodegenerative diseases. Specifically, we will focus on the cellular and molecular mechanisms by which Par-4 sensitizes neurons to apoptosis or necroptosis in different experimental models of AD, including the first mouse “knock-in” model of a naturally occurring presenilin-1 mutation responsible for an early-onset form of Alzheimer disease described by our laboratory. Par-4 significantly increases production of the neurotoxic Aβ species while decreases the release of the neuroprotective sAPP by altering cell death signaling, disrupting intracellular calcium homeostasis, and enhancing amyloidogenic processing of APP. Par-4 also interacts with ACID to promote neurodegeneration in AD by regulating AICD-mediated transcriptional activity. Of importance, we identified AATF (apoptosis antagonizing transcription factor), another leucine zipper domain containing protein, to be an endogenous interaction partner and potent inhibitor of Par-4 activity in neurons. AATF confers neuroprotection by interacting with Par-4 via the leucine zipper domain and interfering with binding of Par-4 to AICD. Of importance, AATF is secreted extracellularly by cortical neurons under neurodegenerative conditions, and secreted AATF (sAATF) blocks TLR4-mediated, RIPK3/MLKL-dependent necroptosis of cortical neurons. Surprisingly, a small core peptide from TRL-4 binding region of AATF, termed as SAP-12, provides a much greater neuroprotective potency and broader effective dose range than the full-length sAATF. Participation of Par-4 in other neurodegenerative diseases and neurological disorders as well as future directions is also discussed.
Alzheimer’s disease (AD) is a neurodegenerative disorder in the central nervous system, and this disease is characterized by extracellular senile plaques and intracellular neurofibrillary tangles. Amyloid-β (Aβ) peptide is the main constituent of senile plaques, and this peptide is derived from the amyloid-β protein precursor (AβPP) through the successive cleaving by β-site AβPP-cleavage enzyme 1 (BACE1) and γ-secretase. AβPP undergoes the progress of post-translational modifications, such as phosphorylation and glycosylation, which might affect the trafficking and the cleavage of AβPP. In the recent years, about 10 phosphorylation sites of AβPP were identified, and they play complex roles in glycosylation modification and cleavage of AβPP. In this article, we introduced the transport and the cleavage pathways of AβPP, then summarized the phosphorylation and glycosylation sites of AβPP, and further discussed the links and relationship between phosphorylation and glycosylation on the pathways of AβPP trafficking and cleavage in order to provide theoretical basis for AD research.
Histone acetyltransferases (HATs) are essential enzymes that are, among others, involved in the epigenetic regulation of gene transcription. They exert their functions through acetylation of both histone and nonhistone proteins and are often part of multiprotein complexes. This chapter mainly discusses recent studies on the functions of HATs in diseases and their utility as potential drug targets. Furthermore, this chapter describes the recent progress in the development of small-molecule HAT modulators and their therapeutic potential. Dysregulation of HAT-mediated acetylation of histone and nonhistone proteins contributes to the pathogenesis and progression of many diseases, such as cancers, inflammatory diseases, and neurological disorders. In recent years, a variety of HAT modulators has been developed, including inhibitors and activators of HAT catalytic activity. Furthermore, inhibitors of HAT protein-protein interactions were developed such as inhibitors of HAT bromodomains. An interesting novel development in HAT-directed drug discovery is the development of proteolysis-targeting chimeras (PROTACs) that trigger proteasome-mediated degradation of HAT proteins. Taken together, these studies demonstrate that HAT proteins are versatile and promising targets for drug discovery.
The scaffolding protein family Fe65, composed of Fe65, Fe65L1, and Fe65L2, was identified as an interaction partner of the amyloid precursor protein (APP), which plays a key function in Alzheimer’s disease. All three Fe65 family members possess three highly conserved interaction domains, forming complexes with diverse binding partners that can be assigned to different cellular functions, such as transactivation of genes in the nucleus, modulation of calcium homeostasis and lipid metabolism, and regulation of the actin cytoskeleton. In this article, we rule out putative new intracellular signaling mechanisms of the APP-interacting protein Fe65 in the regulation of actin cytoskeleton dynamics in the context of various neuronal functions, such as cell migration, neurite outgrowth, and synaptic plasticity.
Persons with Down syndrome (DS, trisomy 21) have widespread cellular protein trafficking defects. There is a paucity of data describing the intracellular transport of IgG in the context of endosomal-lysosomal alterations linked to trisomy 21. In this study, we analyzed the intracellular traffic of IgG mediated by the human neonatal Fc receptor (FcRn) in fibroblast cell lines with trisomy 21. Intracellular IgG trafficking studies in live cells showed that fibroblasts with trisomy 21 exhibit higher proportion of IgG in lysosomes (~ 10% increase), decreased IgG content in intracellular vesicles (~ 9% decrease), and a trend towards decreased IgG recycling (~ 55% decrease) in comparison to diploid cells. Amyloid-beta precursor protein (APP) overexpression in diploid fibroblasts replicated the increase in IgG sorting to the degradative pathway observed in cells with trisomy 21. The impact of APP on the expression of FCGRT (alpha chain component of FcRn) was investigated by APP knock down and overexpression of the APP protein. APP knock down increased the expression of FCGRT mRNA by ~ 60% in both diploid and trisomic cells. Overexpression of APP in diploid fibroblasts and HepG2 cells resulted in a decrease in FCGRT and FcRn expression. Our results indicate that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS.
The amyloid precursor protein (APP) involved in Alzheimer's disease is a member of a larger gene family including amyloid precursor-like proteins APLP1 and APLP2. We generated and examined the phenotypes of mice lacking individual or all possible combinations of APP family members to assess potential functional redundancies within the gene family. Mice deficient for the nervous system-specific APLP1 protein showed a postnatal growth deficit as the only obvious abnormality. In contrast to this minor phenotype, APLP2 ⁻ /− /APLP1 ⁻ /− and APLP2 ⁻ /− /APP ⁻ /− mice proved lethal early postnatally. Surprisingly, APLP1 ⁻ /− /APP ⁻ /− mice were viable, apparently normal, and showed no compensatory upregulation of APLP2 expression. These data indicate redundancy between APLP2 and both other family members and corroborate a key physiological role for APLP2. This view gains further support by the observation that APLP1 ⁻ /− /APP ⁻ /− /APLP2 +/− mice display postnatal lethality. In addition, they provide genetic evidence for at least some distinct physiological roles of APP and APLP2 by demonstrating that combinations of single knock-outs with the APLP1 mutation resulted in double mutants of clearly different phenotypes, being either lethal, or viable. None of the lethal double mutants displayed, however, obvious histopathological abnormalities in the brain or any other organ examined. Moreover, cortical neurons from single or combined mutant mice showed unaltered survival rates under basal culture conditions and unaltered susceptibility to glutamate excitotoxicity in vitro .
A family of highly polymorphic neuronal cell surface proteins, the neurexins, has been identified. At least two genes for neurexins exist. Each gene uses alternative promoters and multiple variably spliced exons to potentially generate more than a 100 different neurexin transcripts. The neurexins were discovered by the identification of one member of the family as the receptor for alpha-latrotoxin. This toxin is a component of the venom from black widow spiders; it binds to presynaptic nerve terminals and triggers massive neurotransmitter release. Neurexins contain single transmembrane regions and extracellular domains with repeated sequences similar to sequences in laminin A, slit, and agrin, proteins that have been implicated in axon guidance and synaptogenesis. An antibody to neurexin I showed highly concentrated immunoreactivity at the synapse. The polymorphic structure of the neurexins, their neural localization, and their sequence similarity to proteins associated with neurogenesis suggest a function as cell recognition molecules in the nerve terminal.
We have isolated a rat cDNA, named FE65, hybridizing to an mRNA of about 2,300 nucleotides present in rat brain, undetectable in rat liver and very poorly represented in other tissues. An mRNA of the same size is present in human neuroblastoma cells and is absent from other human cell lines. The FE65 cDNA contains an open reading frame (ORF) coding for a polypeptide of 499 amino acids in which 143 residues can be aligned with the DNA binding domain of the integrases encoded by mammalian immunodeficiency viruses. The remaining part of the FE65 ORF is not homologous with the correspondent regions of the integrases; the first 206 residues of the FE65 ORF show numerous negative charges and a short sequence not dispensable for the function of the transactivating acidic domain of the jun family transcriptional factors. A plasmid which expresses FE65 amino acids 1-232 fused to the yeast GAL4 DNA binding domain was co-transfected with a plasmid containing five GAL4 binding sites upstream of a minimal Adenovirus promoter controlling the expression of the CAT gene. This experiment showed that the fused protein GAL4-FE65 is able to obtain a 30-40 fold increase of the CAT gene expression compared to the expression observed in the presence of the GAL4 DNA binding domain alone. Two types of FE65 mRNA are present in rat brain, differing only for six nucleotides. We demonstrate that this is the consequence of a neuron-specific alternative splicing of a six-nucleotide miniexon, which is also present in the human genome, in an intron/exon context very similar to that of the rat FE65 gene.
The Tat protein of the human immunodeficiency virus (HIV) is a powerful activator of HIV gene expression. Genetic and biochemical evidence suggests that one or more cellular cofactors may be important for Tat activity. We have used two-hybrid interactive cloning in yeast to identify a partial cDNA clone (clone 10) from a human B-lymphoblastoid library that specifically interacts with the N-terminal 31 amino acids of HIV-1 Tat which contains the essential cysteine-rich portion of the Tat activation domain. The encoded protein also binds to purified Tat in vitro. Mutation of single essential cysteine residues in Tat abolishes interaction between Tat and clone 10, suggesting that interaction with the encoded protein is important for Tat activity. We have identified the full-length cDNA for the Tat binding protein and shown that overexpression of the encoded protein, Tip60 (Tat interactive protein, 60 kDa), results in a fourfold augmentation of Tat transactivation of the HIV-1 promoter in transient expression assays without increasing the basal activity of the HIV promoter or activating the heterologous RSV promoter. These data together with the genetic and in vitro binding data support the notion that Tip60 might be a cofactor of Tat involved in the regulation of HIV gene expression.
The phosphotyrosine interaction (PI) domains (also known as the PTB, or phosphotyrosine binding, domains) of Shc and IRS-1 are recently described domains that bind peptides phosphorylated on tyrosine residues. The PI/PTB domains differ from Src homology 2 (SH2) domains in that their binding specificity is determined by residues that lie amino terminal and not carboxy terminal to the phosphotyrosine. Recently, it has been appreciated that other cytoplasmic proteins also contain PI domains. We now show that the PI domain of X11 and one of the PI domains of FE65, two neuronal proteins, bind to the cytoplasmic domain of the amyloid precursor protein ((beta)APP). (beta)APP is an integral transmembrane glycoprotein whose cellular function is unknown. One of the processing pathways of (beta)APP leads to the secretion of A(beta), the major constituent of the amyloid deposited in the brain parenchyma and vessel walls of Alzheimer's disease patients. We have found that the X11 PI domain binds a YENPTY motif in the intracellular domain of (beta)APP that is strikingly similar to the NPXY motifs that bind the Shc and IRS-1 PI/PTB domains. However, unlike the case for binding of the Shc PI/PTB domain, tyrosine phosphorylation of the YENPTY motif is not required for the binding of (beta)APP to X11 or FE65. The binding site of the FE65 PI domain appears to be different from that of X11, as mutations within the YENPTY motif differentially affect the binding of X11 and FE65. Using site-directed mutagenesis, we have identified a crucial residue within the PI domain involved in X11 and FE65 binding to (beta)APP. The binding of X11 or FE65 PI domains to residues of the YENPTY motif of (beta)APP identifies PI domains as general protein interaction domains and may have important implications for the processing of (beta)APP.
Tip60, originally isolated as an HIV-1-Tat interactive protein, contains an evolutionarily conserved domain with yeast silencing
factors. We demonstrate here direct biochemical evidence that this domain of Tip60 has histone acetyltransferase activity.
The purified recombinant effectively acetylates H2A, H3, and H4 but not H2B of core histone mixtures. This substrate specificity
has not been observed among histone acetyltransferases analyzed to date. These results indicate that Tip60 is a histone acetyltransferase
with a novel property, suggesting that Tip60 and its related factors may introduce a distinct alteration on chromatin.
The Drosophila Notch (N) gene encodes a conserved single-pass transmembrane receptor that transduces extracellular signals controlling cell fate. Here, we present evidence that the intracellular domain of Notch gains access to the nucleus in response to ligand, possibly through a mechanism involving proteolytic cleavage and release from the remainder of the protein. In addition, our results suggest that signal transduction by Notch depends on the ability of the intracellular domain, particularly the portion containing the CDC10 repeats, to reach the nucleus and to participate in the transcriptional activation of downstream target genes.
We have examined the trafficking and metabolism of the beta-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Abeta, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.
Signalling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of gamma-secretase-mediated cleavage of beta-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimer's disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by gamma-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologues in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signalling pathway. Here we report that, in mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signalling pathway that is affected by PS1. Moreover, several gamma-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of gamma-secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signalling.
In the present review, we shall discuss the pros and cons of a possible functional relationship and contribution of the APP family members (APP, APLP1 and APLP2) to the development of Alzheimer's disease: (1) APP, APLP1 and APLP2 are highly homologous proteins with similar protein domain organization. (2) All APP family proteins have been found to be aggregated in typical Alzheimer's disease lesions. (3) Several other proteins have been implied to provide a functional link among the APP-related proteins. In normal adult brain APP, APLP1 and APLP2 are involved in synaptic processes important for memory function. We hypothesize that the functional loss of members of the APP family contributes to the gradual cognitive decline in Alzheimer's disease patients.
The amyloid beta-protein precursor gives rise to the amyloid beta-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid beta-protein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspase-cleaved amyloid beta-protein precursor and activated caspase-9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid beta-protein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease.
The phosphotyrosine-binding (PTB) domain of the cell fate determinant Numb is involved in the formation of multiple protein complexes in vivo and can bind a diverse array of peptide sequences in vitro. To investigate the structural basis for the promiscuous nature of this protein module, we have determined its solution structure by NMR in a complex with a peptide containing an NMSF sequence derived from the Numb-associated kinase (Nak). The Nak peptide was found to adopt a significantly different structure from that of a GPpY sequence-containing peptide previously determined. In contrast to the helical turn adopted by the GPpY peptide, the Nak peptide forms a beta-turn at the NMSF site followed by another turn near the C-terminus. The Numb PTB domain appears to recognize peptides that differ in both primary and secondary structures by engaging various amounts of the binding surface of the protein. Our results suggest a mechanism through which a single PTB domain might interact with multiple distinct target proteins to control a complex biological process such as asymmetric cell division.
In this study we addressed the question of the intracellular localization of Fe65, an adaptor protein interacting with the
β-amyloid precursor protein (APP) and with the transcription factor CP2/LSF/LBP1. By using tagged Fe65 expression vectors,
we observed that a significant fraction of Fe65 is localized in the nucleus of transfected COS7 cells. Furthermore, the isolation
of nuclei from untransfected PC12 cells allowed us to observe that a part of the endogenous Fe65 is present in the nuclear
extract. The analysis of Fe65 mutant constructs demonstrated that the region of the protein required for its nuclear translocation
includes the WW domain, and that, on the other hand, a small fragment of 100 residues, including this WW domain, contains
enough structural information to target a reporter protein (green fluorescent protein (GFP)-GFP) to the nucleus. To evaluate
whether the Fe65-APP interaction could affect Fe65 intracellular trafficking, COS7 cells were cotransfected with APP695 or APP751 and with GFP-Fe65 expression vectors. These experiments demonstrated that Fe65 is no longer translocated to the nucleus when
the cells overexpress APP, whereas the nuclear targeting of GFP-Fe65 mutants, unable to interact with APP, is unaffected by
the coexpression of APP, thus suggesting that the interaction with APP anchors Fe65 in the cytosol.
SNT adaptor proteins transduce activation of fibroblast growth factor receptors (FGFRs) and neurotrophin receptors (TRKs) to common signaling targets. The SNT-1 phosphotyrosine binding (PTB) domain recognizes activated TRKs at a canonical NPXpY motif and, atypically, binds to nonphosphorylated FGFRs in a region lacking tyrosine or asparagine. Here, using NMR and mutational analyses, we show that the PTB domain utilizes distinct sets of amino acid residues to interact with FGFRs or TRKs in a mutually exclusive manner. The FGFR1 peptide wraps around the beta sandwich structure of the PTB domain, and its binding is possibly regulated by conformational change of a unique C-terminal beta strand in the protein. Our results suggest mechanisms by which SNTs serve as molecular switches to mediate the essential interplay between FGFR and TRK signaling during neuronal differentiation.
GAL4, a protein that activates transcription in yeast, is shown to activate the mouse mammary tumor virus promoter in mammalian cells. Activation depends upon a GAL4-binding sequence inserted upstream of the gene. Deletion mutants of GAL4 bearing one or both of the "activating regions" required for activation in yeast also activate transcription in mammalian cells. A derivative of GAL4 that binds to DNA but cannot activate transcription in yeast also fails to activate transcription in mammalian cells. We also show that GAL4 and the glucocorticoid receptor activate the mouse mammary tumor virus promoter synergistically.
We have demonstrated the use of the Escherichia coli LexA repressor-operator system to down-regulate gene expression in mouse cells. The LexA gene was placed downstream of the RSVLTR promoter with polyadenylation and splice signals from SV40. This expression unit was introduced into mouse Ltk- cells by calcium phosphate transfection and stable transfectants selected which express LexA protein. We have used the bacterial chloramphenicol acetyltransferase gene (CAT) as our reporter gene. Transcription of this gene was driven by the HSV tk promoter, into which we have introduced one or two synthetic LexA operator sequences in various positions throughout the promoter. Necessary 3' signals were from the HSV tk gene. Repression by LexA was assessed by comparing the transient expression of tkCAT target constructs, containing LexA operator sequences in the promoter, in cells expressing LexA protein with that in control cells not expressing the repressor. We have observed up to 10-fold repression of CAT expression in LexA+ cells from promoters containing LexA operator sequences.
The secreted form of beta-amyloid precursor protein (APP) containing the Kunitz proteinase inhibitor (KPI) domain, also called protease nexin II, is internalized and degraded by cells. We show that the low density lipoprotein (LDL) receptor-related protein (LRP) is responsible for the endocytosis of secreted APP. APPs770 degradation is inhibited by an LRP antagonist called the receptor-associated protein (RAP) and by LRP antibodies and is greatly diminished in fibroblasts genetically deficient in LRP. APPs695, which lacks the KPI domain, is a poor LRP ligand. Since LRP also binds apolipoprotein E (apoE)-enriched lipoproteins and inheritance of the epsilon 4 allele of the apoE gene is a risk factor for Alzheimer's disease (AD), these data link in a single metabolic pathway two molecules strongly implicated in the pathophysiology of AD.
The beta amyloid peptide which accumulates within the brains of patients with Alzheimer's disease (AD) is proteolytically derived from a precursor protein (beta PP). We established and characterized four stably transformed human neuroblastoma cell lines which conditionally expressed a partial beta PP fusion protein (amino-17 residues+carboxyl-99 residues; S beta C). Conditional expression of S beta C was achieved using a tetracycline-responsive promoter system. Expression of this fusion protein in one of the cell lines resulted in pronounced cytotoxicity. Addition of n6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate and/or fetal bovine serum to the culture medium of this cell line further elevated the level of S beta C expression and enhanced the associated cytotoxicity. Conditioned medium, acquired from cells expressing S beta C, was not cytotoxic. These findings suggest that modulation of beta PP expression and/or metabolism can have cytotoxic consequences. This is the first report of cytotoxic effects mediated by conditional expression of a beta PP derivative. This immortal cell line provides a unique opportunity to screen for complementary DNAs which suppress this toxicity. Such cDNAs could help elucidate the processes underlying S beta C mediated cytotoxicity which in turn could further our understanding of the pathogenesis of AD and could also provide additional candidate genes for various forms of familial AD.
Three synaptic proteins, syntaxin, SNAP-25 and synaptobrevin, were recently identified as targets of clostridial neurotoxins that irreversibly inhibit synaptic vesicle fusion. Experiments searching for membrane receptors for N-ethylmaleimide-sensitive fusion protein (NSF), which has an important role in membrane fusion, revealed an ATP-dependent interaction of the same three synaptic proteins with NSF and its soluble attachment proteins. Thus, two independent approaches identify syntaxin, synaptobrevin and SNAP-25 as components of the synaptic vesicle fusion machinery, but their mode of action is unclear. We have now discovered a brain protein of relative molecular mass 67,000 (67K) which binds stably to syntaxin. Amino-acid sequencing and complementary DNA cloning revealed that the 67K protein is encoded by the mammalian homologue of the Caenorhabditis elegans gene unc-18. In C. elegans, unc-18 belongs to a group of genes defined by mutations with a paralytic phenotype and accumulations of acetylcholine, suggesting a defect in neurotransmitter release. The binding of the mammalian homologue of unc-18 (Munc-18) to syntaxin requires the N terminus of syntaxin whereas that of SNAP-25 involves a more C-terminal sequence. Our data suggest that Munc-18 is a previously unidentified essential component of the synaptic vesicle fusion protein complex.
We have identified proteins that interact with H-Ras using a two hybrid system screen of a mouse cDNA library. Approximately 50% of the clones identified encoded portions of the c-Raf and A-Raf serine/threonine kinases. Overlaps among these clones define a conserved 81 residue region of the N-terminus of Raf as the Ras interaction region. We show that Raf interacts with wild-type and activated Ras, but not with an effector domain mutant of Ras or with a dominant-interfering Ras mutant. Using purified bacterially expressed fusion proteins, we show, furthermore, that Ras and the N-terminal region of Raf associate directly in vitro and that this interaction is dependent on GTP bound to Ras.
Fe65 is a protein mainly expressed in several districts of the mammalian nervous system. The search of protein sequence data banks revealed that Fe65 contains two phosphotyrosine interaction (PID) or phosphotyrosine binding (PTB) domains, previously identified in the Shc adaptor molecule. The two putative PID/PTB domains of Fe65 were used to construct glutathione S-transferase-Fe65 fusion proteins. Co-precipitation experiments demonstrated that the Fe65 PID/PTB domains interacted with several proteins of apparent molecular mass 135, 115, 105, and 51 kDa. The region of Fe65 containing the PID/PTB domains was used as a bait to screen a human brain cDNA library in yeast by the two-hybrid system. Three different cDNA clones were isolated, two of which contain overlapping segments of the cDNA encoding the COOH terminus of the Alzheimer's beta-amyloid-precursor protein (APP), that represents the short intracellular domain of this membrane protein. The third clone contains a cDNA fragment coding for the COOH terminus of the human counterpart of a mouse beta-amyloid-like precursor protein. The alignment of the three APP encoding cDNA fragments found in the screening suggests that the region of APP involved in the binding is centered on the NPTY sequence, which is analogous to that present in the intracellular domains of the growth factor receptors interacting with the PID/PTB domain of Shc.
We have used the yeast two-hybrid system to screen for proteins that interact with the carboxy-terminal domain of APP. Six different clones were isolated and sequence analyses revealed that they encoded domains of a previously described neuronal protein Fe65, a homologue of Fe65 and a homologue of protein X11. All of these proteins contain one or more phosphotyrosine binding (PTB) domains. PTB domain proteins bind to the sequence Asn-Pro-X-Tyr when the Tyr is phosphorylated and are believed to function in signal transduction. APP contains such a motif. These results are consistent with a role for APP in signal transduction mechanisms.
Munc18-1 is a neuronal protein that interacts with syntaxin 1 and is required for synaptic vesicle exocytosis. We have now identified two Munc18-1-interacting proteins called Mint1 and Mint2 that may mediate the function of Munc18-1. Mint proteins are detectable only in brain and are composed of an N-terminal sequence that binds Munc18-1, a middle phosphotyrosine-binding domain, and two C-terminal PDZ domains thought to attach proteins to the plasma membrane. In brain, Mint proteins are part of a multimeric complex containing Munc18-1 and syntaxin that likely functions as an intermediate in synaptic vesicle docking/fusion. The phosphotyrosine-binding domain specifically binds to phosphatidylinositol phosphates known to be produced during vesicle exocytosis (Hay, J. C., Fisette, P. L., Jenkins, G. H., Fukami, K., Takonawa, T., Anderson, R. A., and Martin, T. F. J. (1995) Nature 374, 173-177). Our data suggest a model whereby local production of phosphatidylinositol phosphates may trigger the binding of vesicles to the active zone via the Mint.Munc18-1 complex in conjunction with syntaxin 1.
Notch proteins are ligand-activated transmembrane receptors involved in cell-fate selection throughout development. No known enzymatic activity is contained within Notch and the molecular mechanism by which it transduces signals across the cell membrane is poorly understood. In many instances, Notch activation results in transcriptional changes in the nucleus through an association with members of the CSL family of DNA-binding proteins (where CSL stands for CBF1, Su(H), Lag-1). As Notch is located in the plasma membrane and CSL is a nuclear protein, two models have been proposed to explain how they interact. The first suggests that the two interact transiently at the membrane. The second postulates that Notch is cleaved by a protease, enabling the cleaved fragment to enter the nucleus. Here we show that signalling by a constitutively active membrane-bound Notch-1 protein requires the proteolytic release of the Notch intracellular domain (NICD), which interacts preferentially with CSL. Very small amounts of NICD are active, explaining why it is hard to detect in the nucleus in vivo. We also show that it is ligand binding that induces release of NICD.
The neural protein Fe65 possesses three putative protein-protein interaction domains: one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1 and PID2); the most C-terminal of these domains (PID2) interacts in vivo with the Alzheimer's beta-amyloid precursor protein, whereas the WW domain binds to Mena, the mammalian homolog of Drosophila-enabled protein. By the interaction trap procedure, we isolated a cDNA clone encoding a possible ligand of the N-terminal PID/PTB domain of Fe65 (PID1). Sequence analysis of this clone revealed that this ligand corresponded to the previously identified transcription factor CP2/LSF/LBP1. Co-immunoprecipitation experiments demonstrated that the interaction between Fe65 and CP2/LSF/LBP1 also takes place in vivo between the native molecules. The localization of both proteins was studied using fractionated cellular extracts. These experiments demonstrated that the various isoforms of CP2/LSF/LBP1 are differently distributed among subcellular fractions. At least one isoform, derived from alternative splicing (LSF-ID), is present outside the nucleus; Fe65 was found in both fractions. Furthermore, transfection experiments with an HA-tagged CP2/LSF/LBP1 cDNA demonstrated that Fe65 interacts also with the nuclear form of CP2/LSF/LBP1. Considering that the analysis of Fe65 distribution in fractionated cell extracts demonstrated that this protein is present both in nuclear and non-nuclear fractions, we examined the expression of Fe65 deletion mutants in the two fractions. This analysis allowed us to observe that a small region N-terminal to the WW domain is phosphorylated and is necessary for the presence of Fe65 in the nuclear fraction.
The results summarized in this minireview provide a basic skeleton for the proteolysis model of Notch activation. The challenge for researchers now is to confirm aspects of the model in vivo, to flesh out details of the model, to identify and assign functions to the molecules involved in Notch signaling, and to integrate the model with previous work on Notch signaling. It also remains to be determined whether the proteolysis model is true for Notch signaling in all contexts, particularly those in which Notch signaling is independent of Su(H) or CBF1.
It is a truism of modern biomedical science that the development of therapies expected to slow or arrest the progression of a disease requires as detailed an understanding of its molecular and cellular pathogenesis as possible. In turn, the cloning of novel gene products implicated in a disease often leads to new insights about fundamental features of protein structure and function. A particularly compelling example of this beneficial interplay between basic and applied cell biology arises from the exciting recent progress in deciphering Alzheimer's disease (AD). This review discusses the current understanding of the cell biology of two proteins crucial for the pathogenesis of AD, the beta-amyloid precursor protein and presenilin.
Recent advances in a variety of areas of research, particularly in genetics and in transgenic (Tg)/gene targeting approaches, have had a substantial impact on our understanding of Alzheimer's disease (AD) and related disorders. After briefly reviewing the progress that has been made in diagnostic assessments of patients with senile dementia and in investigations of the neuropathology of AD, we discuss some of the genes/proteins that are causative or risk factors for this disease, including those encoding amyloid precursor protein, presenilin 1 and 2, and apolipoprotein E. In addition, we comment on several potential new candidate loci/genes. Subsequently, we review selected recent reports of analyses of a variety of lines of Tg mice that show several neuropathological features of AD, including A beta-amyloid deposits and dystrophic neurites. Finally, we discuss the several important issues in future investigations of Tg mice, with particular emphasis on the influences of genetic strains on phenotype, especially behavior, and strategies for making new models of neurodegenerative disorders. We believe that investigations of these Tg models will (a) enhance understanding of the relationships between impaired performance on memory tasks and the pathological/biochemical abnormalities in brain, (b) help to clarify pathogenic mechanisms in vivo, (c) lead to identification of new therapeutic targets, and (d) allow testing of new treatment strategies first in mice and then, if successful, in humans with AD.
Presenilin proteins have been implicated both in developmental signalling by the cell-surface protein Notch and in the pathogenesis of Alzheimer's disease. Loss of presenilin function leads to Notch/lin-12-like mutant phenotypes in Caenorhabditis elegans and to reduced Notch1 expression in the mouse paraxial mesoderm. In humans, presenilins that are associated with Alzheimer's disease stimulate overproduction of the neurotoxic 42-amino-acid beta-amyloid derivative (Abeta42) of the amyloid-precursor protein APP. Here we describe loss-of-function mutations in the Drosophila Presenilin gene that cause lethal Notch-like phenotypes such as maternal neurogenic effects during embryogenesis, loss of lateral inhibition within proneural cell clusters, and absence of wing margin formation. We show that presenilin is required for the normal proteolytic production of carboxy-terminal Notch fragments that are needed for receptor maturation and signalling, and that genetically it acts upstream of both the membrane-bound form and the activated nuclear form of Notch. Our findings provide evidence for the existence of distinct processing sites or modifications in the extracellular domain of Notch. They also link the role of presenilin in Notch signalling to its effect on amyloid production in Alzheimer's disease.
Presenilins are membrane proteins with multiple transmembrane domains that are thought to contribute to the development of Alzheimer's disease by affecting the processing of beta-amyloid precursor protein. Presenilins also facilitate the activity of transmembrane receptors of the LIN-12/Notch family. After ligand-induced processing, the intracellular domain of LIN-12/Notch can enter the nucleus and participate in the transcriptional control of downstream target genes. Here we show that null mutations in the Drosophila Presenilin gene abolish Notch signal transduction and prevent its intracellular domain from entering the nucleus. Furthermore, we provide evidence that presenilin is required for the proteolytic release of the intracellular domain from the membrane following activation of Notch by ligand.
Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive gamma-secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters gamma-secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces gamma-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of gamma-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp --> Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 --> TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 --> Ala mutation still inhibited gamma-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and gamma-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase, an autoactivated intramembranous aspartyl protease.
Alzheimer's disease (AD) research has shown that patients with an inherited form of the disease carry mutations in the presenilin proteins or the amyloid precursor protein (APP). These disease-linked mutations result in increased production of the longer form of amyloid-beta (the primary component of the amyloid deposits found in AD brains). However, it is not clear how the presenilins contribute to this increase. New findings now show that the presenilins affect APP processing through their effects on gamma-secretase, an enzyme that cleaves APP. Also, it is known that the presenilins are involved in the cleavage of the Notch receptor, hinting that they either directly regulate gamma-secretase activity or themselves are protease enzymes. These findings suggest that the presenilins may prove to be valuable molecular targets for the development of drugs to combat AD.
We thank our colleagues Utpal Dave and Nick Grishin for helpful discussions and critical review of the manuscript. Our research is supported by grants from the National Institutes of Health (HL20948) and the Perot Family Foundation.
It is well known that histone acetylases are important chromatin modifiers and that they play a central role in chromatin transcription. Here, we present evidence for novel roles of histone acetylases. The TIP60 histone acetylase purifies as a multimeric protein complex. Besides histone acetylase activity on chromatin, the TIP60 complex possesses ATPase, DNA helicase, and structural DNA binding activities. Ectopic expression of mutated TIP60 lacking histone acetylase activity results in cells with defective double-strand DNA break repair. Importantly, the resulting cells lose their apoptotic competence, suggesting a defect in the cells' ability to signal the existence of DNA damage to the apoptotic machinery. These results indicate that the histone acetylase TIP60-containing complex plays a role in DNA repair and apoptosis.
Esa1 is the catalytic subunit of the NuA4 histone acetylase (HAT) complex that acetylates histone H4, and it is a member of the MYST family of HAT proteins that includes the MOZ oncoprotein and the HIV-1 Tat interacting protein Tip60. Here we report the X-ray crystal structure of the HAT domain of Esa1 bound to coenzyme A and investigate the protein's catalytic mechanism. Our data reveal that Esa1 contains a central core domain harboring a putative catalytic base, and flanking domains that are implicated in histone binding. Comparisons with the Gcn5/PCAF and Hat1 proteins suggest a unified mechanism of catalysis and histone binding by HAT proteins, whereby a structurally conserved core domain mediates catalysis, and sequence variability within a structurally related N- and C-terminal scaffold determines substrate specificity.
Tip60 was originally isolated as a Tat interactive protein. It was subsequently shown that Tip60 had histone acetyltransferase (HAT) activity. In studies to understand gene-expression regulation that might involve HAT activity, we PCR-amplified Tip60 from a human heart marathon-ready cDNA library. As a result, we identified an alternatively spliced form of Tip60, Tip60beta (we refer to the previously cloned Tip60 as Tip60alpha). Tip60beta cDNA is slightly smaller than Tip60alpha, and sequencing indicates that there is a deletion of 156 bp in the coding region of the gene. The predicted Tip60beta protein therefore lacks 52 amino acids when compared with Tip60alpha. The Tip60alpha gene is encoded by 14 exons, and Tip60beta is an alternatively spliced form resulting from the exclusion of exon 5 during the splicing process. Exon 5 encodes a proline-rich region that is known to be important for protein-protein interaction. Tip60beta is expressed in a variety of human tissues and cell lines, and the protein is present in both the nucleus and cytoplasm in contrast to Tip60alpha, which is entirely nuclear. The results suggest that Tip60beta may have functions additional to those of Tip60alpha in cells and tissues.
University of Texas Southwestern) K. Beyreuther (Heidelberg Germany) and R. Jahn (Goettingen Germany) for reagents and advice. We are grateful to
- J E Herz
- J Olson
- M Lu
- Lush
Südhof unpublished observations
- X Cao